Itajaí is the largest port in southern Brazil and has one of the nation's highest AIDS incidence rates. Since over 400 truck drivers enter the city daily, they may play a key role in the HIV/AIDS epidemic due to transactions with commercial sex workers (CSWs) and/or substance use. We conducted a rapid assessment to establish the context of HIV vulnerability among truckers and CSWs in Itajaí. Forty three in-depth interviews and eight focus groups were conducted with truckers and CSWs. Two truck-driving routes involving brothels, meeting places and drug-use locations were mapped and field observations were collected. Tapes and field notes were transcribed and analyzed for emerging themes. Truck drivers typically had unprotected sex with several partners, including CSWs and truckstop employees. Both truckers and CSWs had low perceived HIV risk in spite of being engaged in high-risk sex behaviors. Use of alcohol and amphetamine-like drugs was frequent among truckers and appeared to influence unsafe sex practices. Knowledge about amphetamine-related risks was low, as was access to health services and HIV/AIDS behavioral interventions. Interventions, targeting truckers, CSWs and truckstop employees, are needed that traverse cities, states and borders and take into account seasonality, spatial context and workplace conditions.