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Background: Over 95% of penicillin allergy labels are inaccurate and may be addressed in low-risk patients using direct oral penicillin challenge (DPC). This study explored the behaviour, attitudes and acceptability of patients, healthcare professionals (HCPs) and managers of using DPC in low-risk patients., Methods: Mixed-method, investigation involving patient interviews and staff focus groups at three NHS acute hospitals. Transcripts were coded using inductive and deductive thematic analysis informed by the Theoretical Domains Framework., Findings: Analysis of 43 patient interviews and three focus groups (28 HCPs: clinicians and managers) highlighted themes of 'knowledge', 'beliefs about capabilities and consequences', 'environmental context', 'resources', 'social influences', 'professional role and identity', 'behavioural regulation and reinforcement' and a cross-cutting theme of digital systems. Overall, study participants supported the DPC intervention. Patients expressed reassurance about being in a monitored, hospital setting. HCPs acknowledged the need for robust governance structures for ensuring clarity of roles and responsibilities and confidence., Conclusion: There were high levels of acceptability among patients and HCPs. HCPs recognised the importance of DPC. Complexities of penicillin allergy (de)labelling were highlighted, and issues of knowledge, risk, governance and workforce were identified as key determinants. These should be considered in future planning and adoption strategies for DPC., Competing Interests: Competing interests: MTK received research funds from NIHR RfPB, MRC CiC, GCRF, FSA and University of Birmingham outside this work. His department at UHB received educational grants for PracticAllergy Course from ALK Abello, Allergy Therapeutics, MEDA, Thermo Fisher Scientific and other pharmaceutical companies over the years. He is Chair of Equality, Diversity and Inclusion working group of BSACI, co-author for BSACI guidelines on penicillin allergy and an associate editor for Clinical Experimental Allergy. RD delivered paid consultancy services to Baxter medical. He has a salaried position as chief executive of UK Sepsis Trust. RB received funding or honoraria for conference attendance, Advisory boards, lectures and training from Pfizer and Menarini. Senior Editor for JAC-AMR (BSAC journal). NP is a NIHR/ HEE CDRF studying non-allergist penicillin allergy de-labelling in secondary care (Clinical Doctoral Research Fellowship). He is co-lead on the BSAC MOOC on non-allergist penicillin allergy de-labelling (British Society Antimicrobial Chemotherapy Massive Open Online Community). SAM is National Clinical Director for the Blood and Infection Programme of Care, NHS England. JATS has research funding from the NIHR and Wellcome Trust in relation to penicillin allergy. He is a member of the British Society for Allergy and Clinical Immunology allergy working party. He is co-lead on the BSAC MOOC on non-allergist penicillin allergy de-labelling (British Society Antimicrobial Chemotherapy Massive Open Online Community) and a BSAC council member., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)