The actin cytoskeleton influences a wide range of functions in nonmuscle somatic cells, including shape, movement, and interactions with extracellular matrices. The role of actin in mammalian male germ cells, however, particularly during post-testicular development, is not well understood. In this paper, we examine 1) the distribution of 3 actin-regulatory proteins (thymosin beta10, destrin, and a testis-specific actin capping protein) involved in controlling the balance between actin monomers (G-actin) and actin filaments (F-actin), and 2) the distribution and polymerization status of actin in bull spermatozoa during epididymal maturation and following acrosomal exocytosis. Results show that in fixed, permeabilized testicular spermatozoa all 3 regulatory proteins (as determined by binding of specific antibodies) are localized primarily to the acrosomal domain but during epididymal maturation they become confined to the equatorial segment. Following ejaculation, however, they extend back into the acrosomal region. In spermatozoa induced to undergo an acrosome reaction with the calcium ionophore, A23187, further rearrangement occurs with destrin, thymosin beta10, and TS-ACP appearing in the postacrosomal domain. Actin is also found over the acrosome of testicular spermatozoa with both G- and F-actin present, although the 2 forms show slightly different patterns of distribution. Subsequently, actin in the sperm head is largely confined to the equatorial segment until F-actin appears in the postacrosomal domain of acrosome-reacted spermatozoa. This redistribution of actin and actin-regulatory proteins, coupled with changing levels of actin polymerization, suggest a continuing role for actin in both post-testicular sperm maturation and acrosomal exocytosis.