Four pigtailed macaques were inoculated with autologous cells expressing low levels of human immunodeficiency virus type 1 (HIV-1). During the first 10 weeks, infectious virus was recovered from peripheral blood mononuclear cells (PBMCs) and lymph nodes from three of the animals. Subsequently, HIV-1 DNA was frequently detected in uncultured PBMCs from all three animals, and virus was isolated from one of them at weeks 38 and 61. The fourth animal, which was rechallenged at week 10 with cell-free virus isolated from one of the others, never became virus isolation positive, but harbored HIV-1 proviral genomes. These virus infections were accompanied by the development of varied HIV-1-specific humoral immune responses. Antibodies to gp160 were first apparent at week 8 in the three initially infected animals and persisted. The animal from whom virus was isolated at late times also developed persisting antibodies to HIV-1 p24 and gp120. Antibodies to gp120 and gp160 became apparent in the rechallenged animal at 1 week following reinoculation, but they waned with time. In vivo passage of the virus was attempted at week 6. One recipient pigtailed macaque and one recipient cynomolgus monkey failed to become detectably infected following transfusion of virus-positive blood and lymph node cells. The long-term presence of HIV-1-specific antibodies and proviral genomes in these animals, and the recovery of infectious virus more than 1 year following inoculation, are indicative of persistent infection, and confirm previous reports that pigtailed macaques are susceptible to HIV-1.