The proportion of living sperm in semen from six representative mammals was assessed by means of a dual staining technique using the stains SYBR-14 and propidium iodide (PI). SYBR-14, a newly developed fluorescent nucleic acid stain, maximally absorbs at 488 nm and emits at 518 nm when bound to DNA. Microscopic examination revealed that SYBR-14 stained the nuclei of living sperm bright green as determined by simultaneous examination of fluorescence and motility. Conversely, PI stained only nonmotile sperm that had lost their membrane integrity. Sperm from bulls, boars, rams, rabbits, mice, and men were stained and examined through use of fluorescence microscopy. The proportions of living and dead sperm were determined by first staining with SYBR-14 and PI and then assessing stain uptake by flow cytometry. Similar staining patterns were observed in all six mammalian species tested. Three populations of sperm were identified: living--SYBR-14 stained, dead--PI stained, and moribund--doubly stained. The SYBR-14 staining was replaced by PI staining as sperm progressed from living to moribund. The transition from green (SYBR-14) to red (PI) fluorescence started at the posterior region of the sperm head and proceeded anteriorly. The proportions of living and dead sperm in mammalian semen were readily identified through use of dual staining with SYBR-14 and PI and quantified through use of flow cytometry.