The effect of preservation on capacitation status of dog spermatozoa was investigated. Split ejaculates from six dogs were assessed as fresh, chilled for 24 h and rewarmed, and frozen-thawed samples. Capacitation-like status was assessed using the chlortetracycline (CTC)-assay and the measurement of sperm motility patterns using a computer-assisted sperm analyzer. Evaluations were performed on washed spermatozoa immediately after dilution in a Tris-fructose-citrate buffer (TFC) or in canine capacitation medium (CCM), and at 2-h intervals during 8 h of incubation in 5% CO2 in air, at 37 degrees C. Preservation decreased significantly the proportion of uncapacitated spermatozoa. In TFC, at hour 0, chilled-rewarmed and frozen-thawed samples had a significantly lower proportion of uncapacitated, viable spermatozoa than the fresh samples (P<0.05) according to the CTC-assay. The time course of capacitation was accelerated in the preserved samples, compared to the fresh ones. During incubation in CCM, the mean time from hour 0 to when, according to the CTC-assay, the highest proportion of capacitated spermatozoawas present in the samples (time-to-peak), was 4 h for fresh and 2 h for chilled-rewarmed and frozen-thawed samples (P<0.1). The highest values for curvilinear line velocity (VCL) and lateral head displacement (LHD), thought to be descriptive of sperm hyperactivation, were also observed 4 and 2 h after incubation began, in the fresh and the preserved samples, respectively. The difference in time-to-peak for VCL and LHD between fresh, chilled-rewarmed and frozen-thawed semen samples was statistically significant (P<0.02). It can be concluded that based on the CTC-assay and the analysis of motility patterns, capacitation-like changes in dog semen seem to be both initiated and accelerated by the preservation procedures.