The feline immunodeficiency virus (FIV) model of vertical human immunodeficiency virus type 1 transmission was used to explore the timing and tissue tropisms associated with intrauterine lentivirus infection. Cats chronically infected with FIV-B-2542 and their cesarean-derived fetuses and placentas were assayed by polymerase chain reaction and coculture at defined gestational intervals. Prevalence of fetal FIV infection was 0 at 3 weeks, 5% at 5 weeks, 38% at 7 weeks, and 60% at 9 weeks (term). Fetal tissues exhibiting the highest viral tropism were blood mononuclear cells and brain (each containing virus in 60% of FIV-positive fetuses) and thymus (47%). Maternal hematologic and virus load markers did not vary substantially with gestational stage. Therefore, fetal and/or placental maturation may determine the timing of lentivirus transmission. FIV infection prevalence in term fetuses was equivalent to that seen previously in vaginally delivered offspring, suggesting that most vertical FIV transmission occurs late in utero rather than intrapartum.