The motility of human spermatozoa and their ability to penetrate zona-free hamster eggs were examined after dilution of the semen with TES-Tris (TEST) yolk buffer and storage for 48 hours at 2 degrees C to 5 degrees C. Semen samples from 10 fertile donors and 19 infertility patients were studied. More than 65% of the spermatozoa which were initially motile in the TEST yolk buffer remained active after storage. During storage, the mean swimming speed of the sperm declined to approximately 60% of the prestorage value. The percentage of zona-free hamster eggs that were penetrated by spermatozoa from patients and donors increased significantly following 48 hours of storage at 2 degrees C to 5 degrees C. Normal semen and abnormal semen were equally preserved by this storage method. This procedure may be used to ship semen samples by commercial transportation to specialized laboratories for testing. Low temperature storage in the TEST yolk buffer appears to enhance the fertilizing capacity of human spermatozoa in vitro.