Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome integrity of bovine sperm processed in egg yolk-based extender by flow cytometer. SYBR-14 and propidium iodide (PI) enabled the discrimination of sperm cells from egg yolk and debris particles, which was instrumental for the flow cytometric analyses of frozen-thawed bovine sperm, because it implied that washing steps to remove egg yolk were no longer required. In addition, phycoerythrin-conjugated peanut agglutinin (PE-PNA) was used to discriminate acrosome-damaged/reacted sperm cells from acrosome-intact cells. Repeatability was calculated using two processed ejaculates of 10 bulls. Three straws per batch were analyzed in duplicate measurements. Method-agreement analysis between the SYBR-14/PE-PNA/PI and fluorescein isothiocyanate (FITC)-conjugated PNA was performed, with FITC-PNA/PI staining being carried out on 14 frozen-thawed semen samples immediately after thawing and after a 3-h incubation at 37 degrees C. The British Standards Institution repeatability index of the SYBR-14/PE-PNA/PI combination was 2.6%. On average, the FITC-PNA/PI method showed a 6.3% overestimation of the live and acrosome-intact sperm cell subpopulation. In conclusion, the new triple-stain combination is highly repeatable and easy to use in routine application, and it provides a more precise estimate for the rate of sperm cells with intact head membrane and acrosome compared to the generally used and validated FITC-PNA/PI staining.