Washing ram spermatozoa two or four times with Krebs-Henseleit-Ringer solution, to achieve dilution rates of 100- or 10,000-fold, damaged the plasma membranes, acrosomes and mitochondria of some spermatozoa. The proportion of spermatozoa with broken plasma membranes over the acrosome increased with the number of washes. Washing once to achieve a dilution rate of 10-fold was sufficient to remove from spermatozoa all the substrate available for NADPH diaphorase and probably glucose-6-phosphate dehydrogenase, but not quite all the substrate for succinic dehydrogenase. A 10-fold dilution was also sufficient to remove glucose-6-phosphate dehydrogenase from a considerable proportion of spermatozoa, but even three washes to achieve a dilution rate of 1000-fold had little effect on succinic dehydrogenase and NADPH diaphorase activity in spermatozoa. The effects of washing could not be attributed to repeated centrifugation of spermatozoa during the process.