Spermatozoa from four regions of the epididymis and from ejaculated semen were evaluated for their resistance to cold shock, progressive motility, and structural stability. Spermatozoa were incubated at 38 C and their percentage of eosinophilia was compared with that of spermatozoa cooled to 0 C in 2 minutes, 10 C in 12 minutes, or 4 C in 22 minutes. Spermatozoa motility was estimated visually under phase-contrast microscopy and was recorded by cinematography. Structural stability of spermatozoa incubated in 0.05 M sodium borate buffer, 0.035 M sodium dodecyl sulfate (SDS), 0.002 M dithiothreitol (DTT), or SDS+DDT was evaluated by phase-contrast and electron microscopy. The percentage of eosinophilic spermatozoa did not differ among regions of the epididymis and was not increased by cold shock. Cooling ejaculated spermatozoa to 0 C in 2 minutes increased (P < 0.01) the occurrence of eosinophilia (32% vs 73%). Spermatozoa released from the caput or proximal corpus epididymidis into 0.154 M NaCl were not progressively motile; only 4% of the spermatozoa from the distal corpus were motile Cauda epididymal and ejaculated spermatozoa exhibited similar motility (41% vs 47%). Stability of chromatin was greater in spermatozoa from the distal corpus than those from the caput epididymis. Chromatin of spermatozoa from the distal corpus was resistant to 7.5 minutes of SDS+DTT treatment, whereas virtually all spermatozoal nuclei from the caput were decondensed by SDS alone. Tail organelles of spermatozoa acquired stability between the proximal corpus and the cauda epididymidis. All tail organelles of spermatozoa from the proximal corpus were dissolved by 7.5 minutes of SDS treatment, whereas tail organelles of distal corpus epididymal spermatozoa had dissolved after 7.5 minutes of SDS+DTT treatment. Stability of tail organelles in cauda epididymal and ejaculated spermatozoa was similar. Seemingly, equine spermatozoa are infertile until they enter the cauda epididymidis.