The authors present a simple and sensitive culture technique for isolation of Human Immunodeficiency Virus (HIV) from small amounts of heparinized whole blood. This method appeared to be highly sensitive at all stages of disease. Among asymptomatics HIV was isolated from 41.3% of subjects by using peripheral blood mononuclear cells in a standard co-cultivation technique and in 89.6% of cases using whole blood. In the group of AIDS patients, in contrast, HIV isolation rate of the two techniques was the same (94.4%). The presence of detectable p24 Ag in plasma did not affect cultural results and no false positive was observed in the control group. Although further studies are needed to better understand the biological significance of a positive cultural result obtained by this method, HIV isolation from whole blood can be routinely employed, especially when small amounts of blood are available.