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Background: Extended-spectrum β-lactamase Enterobacteriaceae (ESBL-E) may be sexually transmitted. Men who have sex with men (MSM) have different sexual behaviour than the general population, and thus may be at risk for ESBL-E carriage. This study determined the prevalence of ESBL-E carriage and its association with sexual behaviour among MSM in Amsterdam, The Netherlands., Materials and Methods: In total, 583 HIV-positive and HIV-negative MSM from the Amsterdam Cohort Study were screened for rectal ESBL-E carriage between April and December 2018. Participants completed a self-administered questionnaire on (sexual) behaviour and risk factors for antimicrobial resistance. The proportion of the study population with ESBL-E carriage was compared by number of sexual partners using logistic regression, and across clusters of sexual behaviours with steady and casual partners, separately, using latent class analyses; all results were adjusted for recent use of antibiotics, travel and hospitalization., Results: Overall, 16.3% [95% confidence interval (CI) 13.4-19.5] of the study population tested positive for ESBL-E. The odds of ESBL-E carriage increased as number of sexual partners increased [adjusted odds ratio per ln(partner+1), 1.57, 95% CI 1.26-1.94; P<0.001]. There was no association between ESBL-E carriage and sexual behaviour with steady partner(s). Compared with participants in the 'no sex with casual partner(s)' cluster, adjusted odds of being ESBL-E positive were 2.95-fold higher (95% CI 1.52-5.80) for participants in the 'rimming and frottage' cluster (P=0.001) and 2.28-fold higher (95% CI 0.98-5.31) for participants in the 'toy use and fisting' cluster (P=0.056)., Conclusions: The prevalence of ESBL-E in MSM is higher compared with the overall Dutch population, likely due to sexual transmission with casual partners. This implies that sexually active MSM should be considered a risk group for ESBL-E carriage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)