SummaryA prospective study was conducted on 61 unselected semen samples from infertile patients to compare the efficacy of human follicular fluid (hFF) or TEST‐egg yolk medium treatment in enhancing sperm performance in the zona‐free hamster oocyte penetration (HOP) test. Both hFF and TEST‐egg yolk enhanced penetration scores significantly in the HOP test in comparison with the control swim‐up sperm preparation. The TEST‐egg yolk medium treatment, however, exhibited the highest penetration scores, and there were higher percentages of preparations passing the 10 or 20|X% penetration rate cut‐off point with sperm exposed to the TEST‐egg yolk medium than those exposed to hFF. These results indicate that modification of the original HOP‐test protocol by incorporation of TEST‐egg yolk medium gives a greatly increased penetration rate when compared to the addition of hFF