The aim of this study was to compare the standard seminal fluid analysis (SFA) with the in vitro sperm penetration test (SP test). Semen from husbands in 100 infertile couples, from 25 fertile donors, and from 25 infertile males with known sperm pathology was examined. In the group of infertile males all of the SP tests were classified as poor. In the group of fertile donors the incidences of poor, inconclusive, and normal SP tests were 8%, 12%, and 80%, respectively. In the group including both normal and infertile male partners, there were 31% poor, 19% inconclusive, and 50% normal SP tests. Comparison of SP tests with different parameters of the SFA revealed that in vitro sperm penetration is mainly influenced by sperm motility and morphology. Sperm count had a limited bearing upon the in vitro penetrating ability of spermatozoa. Among 58 semen samples showing poor mucus penetration in vitro, 39 had both reduced motility and increased concentration of abnormal spermatozoa. On the other hand, this combination of pathologic parameters was not encountered at all among the 92 sperm samples with intermediate or normal sperm penetrating ability. A normal sperm concentration did not necessarily mean good penetration ability. Of 52 samples with a sperm count of more than 60 million/ml, only 53% showed normal sperm penetration.