Since Yanagimachi et al. (1976) suggested that human spermatozoa were capable of penetrating into zona-free hamster eggs, this in vitro assay has been used to analyse the fertilizing ability of human spermatozoa. Serum albumin is an important constituent of the medium used for the assay. However, a great variation in the rate of sperm penetration was observed in the use of different albumin preparations at different concentrations. Therefore we examined the effects of three different kinds of albumin preparations on the rate of human sperm penetration into zona-free hamster eggs. The results obtained were as follows. The percentages of eggs being penetrated by spermatozoa from three fertile donors A,B, and C, were assessed. When Fraction V, Globulin Free and Fatty Acid Free albumin preparations were tested at a concentration of 3.5% (W/V) by the assay using sperm from donor A, penetration rates were 13.3%, 97.4%, and 8.7% respectively. Dilution of the albumin concentration to 0.3% considerably changed the penetration rates to 64.4%, 78.8% and 12.1% in that order. In cases B and C, penetration rates showed the same tendency as in case A. alpha 1 and alpha 2 globulin fractions contaminated in the Fraction V preparation possibly inhibit the human sperm penetration into zona-free hamster eggs. An appropriate quantity of fatty acid is necessary for human spermatozoa to penetrate into zona-free hamster eggs, because penetration rates were low in the percentage of Fatty Acid Free albumin regardless of the concentration added. It is concluded that use of the same preparation of good quality is mandatory for human sperm penetration tests using zona-free hamster eggs to evaluate the results with reproducibility and accuracy.