The objective of this study was to investigate the frequency of HIV infection among female sex workers in the port area of Imbituba (State of Santa Catarina), and to identify the viral subtype and its susceptibility to antiretroviral medications. Ninety women were interviewed between December 2003 and February 2004. Six (6.7%) were HIV-positive. Genotyping for HIV, performed on four samples, detected subtype C in three of them, which is predominant in Africa and Asia, and subtype B in one of them, which is prevalent in Brazil, USA and Europe. The results suggest that the Port of Imbituba may be one of the gateways for HIV-1 subtype C to enter Brazil, and for its dissemination to the rest of the country and the Mercosul area, along the highway BR-101. This points towards the need for preventive work to reduce the introduction and dissemination of HIV subtype C in Brazil.