A study of DNA denaturation patterns in human sperm was conducted as part of a NIOSH longitudinal study of semen quality in males not occupationally or environmentally exposed to toxic chemicals. The study group consisted of 45 males with no known exposures to industrial toxicants. Semen samples were obtained monthly for 8 months after abstinence periods of 0.5 to 11 days. The samples were stained by acridine-orange and the sperm chromatin structure was evaluated by the flow cytometric sperm chromatin assay (SCSA). The SCSA determined the stability of the sperm chromatin structure within a donor over time by measuring the resistance of the sperm DNA to in-situ denaturation. Staining patterns of native and denatured DNA (cytograms) were constructed from the data. The cytograms for each subject were generally unique and homogeneous over time. The cytogram patterns of some subjects showed inter monthly fluctuations but then returned to the original pattern. This was attributed to some type of testicular stress. The overall mean value of a parameter representing the proportion of sperm with denatured DNA (alpha-t) was 222.3. The mean between and within donor standard deviations in alpha-t were 38.9 and 21.8, respectively. The intraclass correlation, a parameter that measured repeatability, for the mean and standard deviations in alpha-t ranged from 0.67 to 0.90. The intraclass correlation was higher than those of conventional sperm quality variables measured in the larger study. Among the conventional semen variables, the SCSA correlated best with percentage sperm swollen in hypoosmotic medium. The SCSA showed low, but statistically significant correlations with sperm concentration, count, and morphology. The authors conclude that the SCSA should be suitable for toxicology field studies of semen quality. The SCSA should be particularly useful for identifying defective samples that appear normal by other semen quality criteria.