The t complex in the mouse is a large group of linked genes that affect sperm function in fertilization. In t/+ males, sperm carrying the t complex (t sperm) have normal fertilizing ability, while sperm carrying the normal homolog (+t sperm) are dysfunctional (P. Olds-Clarke and B. Peitz, 1985, Genet. Res. 47, 49). The specific step in fertilization which is dysfunctional, however, is not known. Two characteristics of fertilizing sperm, capacitation (the process by which sperm become capable of undergoing the acrosome reaction) and hyperactivation (the change in swimming behavior concomitant with capacitation), were assayed by objective methods in epididymal sperm from tw32/+ males of two strains, and compared to sperm from +/+ males of the same strains. Capacitated and acrosome-reacted sperm were identified by a chlortetracycline assay (C.R. Ward and B.T. Storey, 1984, Dev. Biol. 104, 287). Hyperactivated sperm were identified by their path shape and swimming speed, using a computer-assisted motion-analysis system (J.M. Neill and P. Olds-Clarke, 1987, Gamete Res. 18, 121). Hyperactivation occurred significantly sooner among sperm from tw32/+ mice than among sperm from +/+ mice of the same strain, while the rates and maximal levels of capacitation and spontaneous acrosome reactions were normal. Of the nonhyperactivated motile sperm from tw32/+ mice, almost all were slower than sperm from +/+ mice of the same strain. While the effect of premature hyperactivation on fertilization is not clear, slow movements are likely to impair fertilizing ability. These results raise the possibility that the slow sperm are the dysfunctional +t sperm.