In Japanese black cattle, AI severely subfertile males have occasionally been found. In order to solve this problem, we previously asserted the need for exact examinations of acrosomal tyrosine-phosphorylated proteins and acrosome morphology in cryopreserved spermatozoa. In the present study, we further investigated acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation and examined possible relationships between these phosphoproteins and acrosome stability. Ejaculated, epididymal and cryopreserved spermatozoa were subjected to examinations of general characteristics (motility, shape and acrosome morphology) and indirect immunofluorescence of acrosomal phosphoproteins. Unlike all general characteristic parameters, the distribution of acrosomal tyrosine-phosphorylated proteins in ejaculated and cauda epididymal spermatozoa varied considerably among bulls and was linked to the maintenance of morphologically normal acrosomes in cryopreserved spermatozoa or ejaculated spermatozoa after 270min incubation. Moreover, the distribution of these phosphoproteins was arranged in the spermatozoa of the proximal epididymides. These findings indicate that acrosomal tyrosine-phosphorylated proteins are distributionally arranged during early process of sperm maturation, that their distribution of cauda epididymal and ejaculated spermatozoa are largely different among bulls, and that varied states of acrosomal phosphoproteins may result in individual differences in acrosome stability among bulls.