The aim of the study was to determine the risk factors for acquiring brucella infection among healthcare workers (HCWs). The study was performed in Ankara Numune Education and Research Hospital, Turkey, before the introduction of Biosafety III measures. A questionnaire was given to HCWs, who were at risk of brucella infection. Twelve HCWs with brucella infection were detected, an incidence of 8% per employee-year. A multivariate analysis of risk factors in seven of the cases and 48 control HCWs was performed. Male physicians were found to be associated with a higher risk of acquiring brucella infection [odds ratio, 25.3; confidence interval (CI), 2.3-283.7; P=0.008]. Using gloves was found to be protective (odds ratio, 0.02; CI, 0.008-0.4; P=0.017).