The ATP content of bovine semen was measured using a bioluminescent technique. No ATP was detected in seminal plasma or diluent. The mean concentration of ATP (+/- sd) in ejaculated semen was 76.5+/-38.21 n moles ATP/10(8) spermatozoa and in diluted, frozen and thawed semen 10.00+/-4.74 n moles ATP/10(8) spermatozoa. The observed motilities of ejaculated and of diluted, frozen and thawed semen were linearly related to the ATP content r=0.652; p<0.01 and r=0.466; p<0.001 respectively). The mean ATP content and motility calculated for each bull were similarly related (r=0.857; p<0.02 and r=0.607; p<0.05). The technique shows promise in providing an objective measure of spermatozoan activity.