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Objective: To assess the availability and price of naloxone as well as pharmacy staff knowledge of the standing order for naloxone in Pennsylvania pharmacies., Methods: We conducted a telephone audit study from December 2016 to April 2017 in which staff from Pennsylvania pharmacies were surveyed to evaluate naloxone availability, staff understanding of the naloxone standing order, and out-of-pocket cost of naloxone., Results: Responses were obtained from 682 of 758 contacted pharmacies (90% response rate). Naloxone was stocked (ie, available for dispensing) in 306 (45%) pharmacies surveyed. Of the 376 (55%) pharmacies that did not stock naloxone, 118 (31%) stated that they could place an order for naloxone for pickup within 1 business day. Responses by pharmacy staff to questions about key components of the standing order for naloxone were collected from 581 of the 682 pharmacies who participated in the survey (85%). Of the 581 pharmacy staff members who stated that they either stocked or could order naloxone, 64% correctly answered all questions pertaining to understanding of the naloxone standing order. The respective median out-of-pocket prices stated in the audit varied by formulation and ranged from $50 to $4000. Staff from national pharmacies were significantly more likely than staff from regional/local chain and non-chain pharmacies to correctly answer that a prescription was not required to obtain naloxone (68.5%, 57.7%, and 52.4% respectively, (P = 0.0045)., Conclusions: Multiple barriers to naloxone access exist in pharmacies across a large, diverse state, despite the presence of a standing order to facilitate such access. Limited availability of naloxone in pharmacies, lack of knowledge or understanding by pharmacy staff of the standing order, and variability in out-of-pocket cost for this drug are among these potential barriers. Regulatory or legal incentives for pharmacies or drug manufacturers, education efforts directed toward pharmacy staff members, or other interventions may be needed to increase naloxone availability in pharmacies.