In the presence of specific antigen, lymph node cells from Rockefeller strain guinea pigs with delayed hypersensitivity to homologous spermatozoal antigen produced progressive destruction of L929 cell monolayers. This effect was measured by counting surviving target cells removed from Leighton tubes by trypsin treatment as well as by standard cell counts in microplates. The cytotoxic effect was immunologically specific since neither sperm‐sensitized lymphocytes in presence of unspecific antigen nor normal lymph node cells with guinea pig sperm, induced target cell destruction. The effect required an antigen concentration equal to, or greater than, 70 μg protein/ml. A ratio of 100 : 1 of sensitized lymphoid cells to target cell was found to be optimal. L‐cell lysis could be demonstrated at day 14 and reached its peak on day 21 after immunization. A high level of correlation between lymphocyte cytotoxicity and macrophage migration inhibition was found. The role of lymphocyte cytotoxicity in cell‐mediated immunity to spermatozoa is discussed as one of the possible mechanisms leading to destructive changes in the testes.