In 2002-2005, a cross-sectional study to assess the potential for HIV transmission was carried out among 557 female and male-to-female transgender commercial sex workers (CSW) in three cities in the Netherlands. Female CSW (F-CSW), drug-using female CSW (DU), and transgender sex workers were recruited in street-based and establishment-based sites. An anonymous questionnaire was administrated by interviewers and a saliva sample was collected for HIV antibody testing. The overall HIV prevalence was 5.7% (31/547; 10 samples were excluded because of "intermediate" test results). HIV was more prevalent among transgender (18.8%, 13/69) and DU (13.6%, 12/88) sex workers than among F-CSW (1.5%, 6/390). Of the HIV positive CSW, 74% were unaware of their infection. Consistent condom use with clients was 81%. Regular condom failure with clients was reported by 39%. In multivariate analyses, transgender sex workers (OR = 22.9), drug-using CSW who ever injected drugs (OR = 31.1), African (OR = 19.0), and South European ethnicity (OR = 7.2) were independently associated with HIV. Condom failure (PRR = 2.0), anal sex (PRR = 2.1), and drug use (PRR = 3.8) were associated with inconsistent condom use with clients. There is a potential risk for further spread of HIV, through clients and (private) partners of sex workers, to the general population. Targeted health promotion activities are indicated for transgender sex workers and drug-using female CSW; active HIV testing must be continued.