Human endogenous retrovirus K10 (HERV-K10) env and gag expression has been detected in placenta, embryonic tissue, and cell lines. By transfection, these sequences have been expressed in insect cells and developed into serological assays, revealing HERV-K10 antibodies in patients with testicular cancer. Patients with AIDS are at an increased risk for testicular cancer and frequently reactivate latent infections. We postulated that HERV-K10 seroprevalence might be increased with HIV infection or AIDS. Stored, frozen serum samples from 52 patients with testicular cancer (8 patients with HIV and 30 patients with samples near the time of diagnosis) and 84 controls (40 patients with HIV) were diluted 1:40 and tested by immunofluorescence against SF158 cells transfected with HERV-K10 env [ENV1.9(+)] or gag (pACGAG). Seroprevalence rates were compared cross-sectionally in cases and controls, excluding those with indeterminate results (3 of 30 cases and 7 of 84 controls), and also were examined longitudinally in the cases before or after diagnosis of testicular cancer. Seroprevalence to HERV-K10 Env or Gag was 17 of 27 testicular cancer patients (63%) around the time of diagnosis, compared to 4 of 77 controls (5%; P < 0.0001). Seroprevalence was similar (50% to 60%) with seminoma, teratocarcinoma, or embryonal carcinoma, and it was not increased with HIV infection in either cases (33%) or controls (3%). HERV-K10 antibodies were detected in 12 of 19 cases (63%) more than 6 months before seminoma diagnosis, as well as in four cases with residual or recurrent malignancy more than 1 month after initial diagnosis. Thus, HERV-K10 antibodies are detected frequently with testicular cancer and seem to resolve rapidly with effective therapy of the malignancy. Antibody reactivity also occurs in approximately 5% of controls, perhaps because of nonspecific or cross-reactive epitopes. HIV and AIDS were not associated with HERV-K10 antibodies, thus, leaving their higher risk of testicular cancer unexplained.