We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.