2-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and morphological characteristics. In the rat, degenerating spermatocytes appear necrotic at 24 hr, while in the guinea pig they appear apoptotic 96 hr after the start of three daily doses. To further examine if the spermatocyte degeneration in both species represented necrosis or apoptosis, the extent and nature of nuclear DNA fragmentation after ME exposure were assessed both visually using an in situ nucleotide 3' end-labeling (ISEL) procedure and by DNA gel electrophoresis. Testes from rats given a single oral dose of ME (200 mg/kg) showed the expected pachytene spermatocyte degeneration 24 hr after dosing, with the nuclear chromatin degradation typical of necrosis. In contrast, testes from guinea pigs given daily oral doses of ME (200 mg/kg) showed spermatocyte degeneration at only 96 hr after the start of dosing, with marked peripheral nuclear chromatin condensation characteristic of apoptosis. Coincident with the appearance of morphologic changes, degenerating spermatocytes in both species contained fragmented DNA as revealed by the ISEL procedure. The pattern of DNA fragmentation on agarose gels in both species consisted of ordered multiples or "ladders" of approximately 200 base pairs, a hallmark of apoptosis, with their appearance coincident with the time course of morphologic spermatocyte degeneration and ISEL staining. Preliminary data reveal the appearance of divalent metal cation-dependent endonuclease activity at pH 7.0 in ME-treated immature (24-day-old) rat testis that produces a similar pattern of DNA fragmentation and which appears to be distinct from activity associated with the spontaneous germ cell degeneration observed in testes of this age. In summary, in vivo ME exposure induces spermatocyte apoptosis in both the rat and guinea pig despite differing morphological classifications and time of onset of cell death. Future studies will focus on further characterization of the testicular endonuclease in the rat and the potential role of increased intracellular Ca2+ as a "triggering" stimulus in ME-induced spermatocyte apoptosis.