Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.