The objective of this in vitro study was to examine the response of mixed cultures of Sertoli and germ cells to treatment with thallium (Tl) at the range of concentrations that, in previous studies, was shown in vivo to affect reproduction. Cultures were prepared from the testis of Sprague‐Dawley rats. Cultures containing approximately 3.75 × 105cells/ml were treated with Tl concentrations corresponding to 35, 7, and 1.4 μg Tl/g testis, estimated from protein content of cultures. Observations at 24, 48, and 72 h after treatment showed a significant release of germ cells into the culture medium that was both concentration and time dependent. Cultures treated with 35 μg Tl/g testis showed a threefold increase in germ‐cell detachment compared with controls after only 24 h of exposure. As the treatment time increased to 48 h of exposure, even cultures exposed at the lowest Tl concentration (1.4 μg Tl/g testis) showed significant loss of germ cells. After 48 h, cultures exposed to 7 μg Tilg testis exhibited a 2.5‐fold increase in germ‐cell detachment, and those exposed to 35 μg Tl/g testis exhibited a 10‐fold increase over controls. Morphological investigations of cell cultures showed evident loss of germ cells with significant reduction in prepachytene and pachytene spermatocytes and changes in the shape of Sertoli cells. These results are in agreement with in vivo studies, in which thallium treatment at comparable exposure levels manifested its earliest toxic testicular effects in Sertoli and germ cells. They also demonstrate the usefulness of this in vitro culture technique to assess toxic testicular damage rapidly.