Since the description of human T-cell leukemia virus type I (HTLV-I) and its simian counterpart, simian T-cell leukemia virus type I (STLV-I), the possible existence of other related simian retroviruses has been raised. Here, we report a new retrovirus, STLV-II, which we have identified in spider monkeys (Ateles fusciceps), a New World primate species. Initially, a recombinant HTLV-II envelope protein (RP-IIB) was used to identify anti-STLV-II antibodies in New World monkeys by Western blot (immunoblot) assays. Subsequently, the virus was characterized by Southern blot hybridization, which showed that STLV-II and HTLV-II have a high degree of nucleotide sequence homology but have different restriction enzyme patterns. Nucleotide sequence analysis of the pX-II region of STLV-II provirus revealed 3% variation with the corresponding region of HTLV-II. Electron micrographic studies revealed HTLV-like, type C retrovirus particles outside the cell membranes of STLV-II-infected cells. This study describes the first link between HTLV-II and a simian reservoir in the New World. Further molecular studies of STLV-II infection in different species of New World monkeys, especially from the wild, may provide valuable information about the origin and intragroup relationships of South American monkeys. Spider monkeys infected with STLV-II may serve as an important animal model for HTLV-II infection in humans.