The manner in which health care workers (HCWs) interact with HIV/AIDS patients and drug users during their work clearly influences the sustainability of harm reduction programs. To evaluate the professional attitudes of HCWs, we designed a questionnaire with four constructs - discrimination, acceptance of HIV/AIDS patients, acceptance of drug users, and fear - and tested its reliability and validity. Ten experts rated the questionnaire and the mean content validity index was 85.6%. Analysis of 251 anonymous questionnaires from HCWs in Taiwan yielded a composite reliability and Cronbach's α for the four constructs of >0.7. First-order and second-order confirmatory factor analysis revealed a χ(2)/degrees of freedom <3, goodness-of-fit index (GFI) >0.9, adjusted goodness-of-fit index (AGFI) >0.9, Bentler-Bonnett normal fix index >0.9, and a root mean square error of approximation between 0.00 and 0.07 indicating a good fit of the model. HCWs with training in HRPs or AIDS prevention had higher questionnaire scores than those without such training, indicating good known-group validity.