Sperm cryopreservation and thawing reduces fertility and alters the content and function of various sperm proteins. Previously, we reported that a testes-specific isoform of angiotensin-converting enzyme (tACE) was required for capacitation of bovine spermatozoa. The aim of the present study was to determine effects of sperm cryopreservation and thawing on the content, activity and localisation of tACE in bovine spermatozoa. Relative median fluorescence intensity (flow cytometry) was greater (P<0.01), tACE content (110 kDa protein) in sperm proteins was higher (P<0.01) and there was greater tACE enzyme activity (mean (±s.e.m.) 0.16±0.01 vs 0.06±0.02UmL-1; P<0.01) in fresh versus frozen-thawed spermatozoa (n=6 bulls). In fresh spermatozoa, tACE was immunolocalised in the acrosomal and principal piece regions of the sperm head and tail respectively. However, in frozen-thawed spermatozoa, there were four patterns of localisation: most frozen-thawed spermatozoa (64%) had fluorescence in the acrosomal ridge, whereas in 17% and 9% of spermatozoa the signal was limited to the post-acrosomal region and the equatorial segment respectively; in the remainder (10%), there was no signal. We conclude that cryopreservation and thawing decrease the content and activity of tACE and cause it to be translocated to other parts of the sperm head.