Cell associated and cell-free simian immunodeficiency virus (SIV) were used to investigate transmission of SIV across the vaginal mucosa of rhesus macaques. The intact vaginal epithelium was found to be a strong but penetrable barrier to cell-free SIV infection. We found that 10,000-fold more cell-free SIV was needed to infect 100% of the macaques by the vaginal route when compared to the dose needed to infect 100% by the intravenous (iv) route. Like cell-free SIV, cell-associated SIV was an efficient means of transmission if given by the iv route; as few as 2 SIV-infected peripheral blood mononuclear cells (PBMC) were infectious inoculum. However, macaques were resistant to cell-associated SIV when exposed by the vaginal route; 10,000 SIV-infected PBMC failed to infect vaginally inoculated macaques. It was also found that vaginal transmission of cell-free SIV to macaques increased during the luteal phase of the menstrual cycle compared to the follicular phase. Results with this animal model predict that cell-free human immunodeficiency virus (HIV) is likely to be the more efficient mode of HIV vaginal transmission and that susceptibility may vary during the menstrual cycle.