We used the polymerase chain reaction on 63 tissue specimens of histologically staged classic Kaposi's sarcoma (KS) from 40 patients, 14 specimens from 14 acquired immune deficiency syndrome (AIDS)-KS cases (all from the same geographic area over a 10-year period), and peripheral blood mononuclear cells from 1 of the non-AIDS KS patients to amplify a specific 210-bp genomic sequence of the newly discovered KS-associated herpesvirus (KSHV). Also tested were 86 benign and malignant endothelial lesions, which potentially simulated each KS histological stage and were further matched by age approximation and by sex with a classical KS specimen. The lesions included hemangioma, lymphangioma, pyogenic granuloma, and angiosarcoma. KSHV was also sought in multiple well characterized vascular endothelial cell lines from AIDS-KS lesions and in 20 mainly cutaneous benign and malignant lesions from 15 immunosuppressed transplant patients. Overall, 92% of KS tissue specimens, representing 88% of classical KS and 100% of AIDS-KS patients, and in addition the sample of peripheral blood mononuclear cell DNA, were positive as visualized on ethidium bromide gels and confirmed by Southern blot hybridization (only 1 case was negative on gell visualization but positive on Southern blot), thus confirming the close association of KSHV with KS of different clinical forms. None of the various other endothelial lesion, skin lesions in immunosuppressed patients, or AIDS-KS endothelial cell lines contained amplifiable KSHV DNA, which indicates that reactivation of KSHV is not present in the skin lesions of immunosuppressed patients and probably is not a ubiquitous agent that secondarily infects proliferative endothelium. The absence of amplifiable virus DNA in the cultured endothelium of KS suggests that the stimulus for angioproliferation originates in another host cell or under conditions not reproduced in culture. The polymerase chain reaction is a specific and sensitive means of verifying KS in the differential diagnosis of angioproliferative lessons.