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The relationship between viral infection with Kaposi sarcoma-associated herpesvirus (KSHV) and the onset of Kaposi sarcoma (KS) in AIDS patients is incompletely understood. This study investigates the use of three serological assays to predict the development of KS in HIV-positive patients. Serially collected serum samples from 36 patients with KS and matched controls in the Swiss HIV Cohort Study (SHCS) were analyzed in a case control study. Three serologic assays to detect antibodies against KSHV (nuclear and membrane antigen immunofluorescence assay, N-IFA, M-IFA and ORF 65.2 ELISA) were used to determine the predictive value of KSHV-seropositivity. Serial samples from the cases were also analyzed to determine longitudinal patterns of seroreactivity and identify cases of seroconversion. Assay sensitivity for detection of KSHV antibodies was highest for M-IFA (83%), followed by N-IFA (74%) and 65.2 ELISA (52%). At the time of initial serum sampling (median 4.7 years before KS), only the N-IFA distinguished case and control sera (61% vs. 32%) and no assay was clearly predictive of subsequent onset of clinical KS. Moreover, an unexpectedly high rate of reversions to seronegativity were observed by N-IFA (27/33) as well as by 65.2 ELISA (11/26) in the longitudinal analysis. Analysis of the ORF65.2 ELISA index indicated that these reversions before the clinical onset of KS were associated with antibody levels that frequently hovered around the level of detectability. A marked increase in ORF 65.2 antibody titer occurred in a third of the patients at the time of KS diagnosis. Only two seroconversions were documented. KSHV infection within the SHCS is likely to have preceded HIV infection. KSHV infection alone is not highly predictive of KS development in this cohort of HIV-infected homosexual men as compared with matched controls. Three KSHV serologic assays, though sensitive at the time of clinical KS are inconsistently positive before the development of AIDS-related KS.