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Three methods were used to prepare spermatozoa for subzonal injection into mature oocytes. In method A, the washed sperm suspension was incubated for 18 h in modified T6 culture medium. Method B consisted of incubating the sperm suspension for 6 h in regular T6 culture medium. In method C, the sperm suspension was incubated for 6 h in regular T6 culture medium containing 20% (v/v) follicular fluid. The percentages of acrosome-free spermatozoa and fertilization rates were compared for 42 treatment cycles assigned randomly to the three sperm preparation methods. The sperm suspensions prepared by methods A and C each had significantly higher proportions of acrosome-free spermatozoa compared to suspensions prepared by method B. The fertilization rates of oocytes micro-injected with spermatozoa prepared by methods A and C were significantly higher than for method B. Eight clinical pregnancies resulted from 28 cycles in which embryo replacement occurred. We conclude that the fertilization rate following subzonal sperm injection is related directly to the percentage of acrosome-free spermatozoa in the sperm suspension used for microinjection.