The sperm chromatin structure assay (SCSA), as developed by Evenson et al.(1980), utilizes flow cytometry to quantify the susceptibility of sperm chromatin to in situ acid denaturation via the metachromatic properties of acridine orange. SCSA is repeatable and has been used to distinguish between fertile and subfertile males in different species; however, it does not permit morphological evaluation of cells. In the present study, the SCSA was modified for the fluorescence/differential interference contrast (DIC) microscope to examine morphology and chromatin stability on the same cell. Semen from six Holstein bulls was collected twice weekly for six weeks. Semen was cryopreserved after collection. A 48-hr scrotal insulation was applied after the first three collections to exert a mild thermal insult to the testes; this induces specific spermatozoal morphological abnormalities to appear in a predictable chronological order, as determined by Vogler et al. (1993). Using DIC optics, sperm head morphology was classified as normal, slightly misshapen, pyriform, severely misshapen, or tailless. Vacuolization in the head region was scored separately as apical, diadem, or random. SCSA and modified-SCSA for fluorescence microscopy were used to assess chromatin instability in the samples. The SCSA parameter of 'cells outside the main population of alpha t' (%COMP alpha t) and the modified-SCSA parameter of '% cells shifted from green' were positively correlated (r=0.84; P<0.01). Both variables were positively correlated with the appearance of tailless, pyriform, severely misshapen, and randomly vacuolated cells (P< 0.01), but not with the appearance of diadems or apical vacuoles. Also, the fluorescence microscope detected a significant shift from green in normally shaped cells appearing in morphologically abnormal ejaculates (P<0.01). These results demonstrate that scrotal insulation-induced morphological abnormalities in spermatozoa signify a perturbation in chromatin structure, and that the chromatin perturbation extends into normally shaped cells in the same ejaculate.