The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 degrees C for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 degrees C for 45 min and -30 degrees C for 30 min, and thawed at 50 degrees C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.