Motility and energy level in sperm cells are tightly linked, but not totally understood. The aim of this study was to examine whether adenosine triphosphate (ATP) content as a sperm quality parameter for bull semen could give additional information together with viability and motility. The objective was therefore to examine the relationships between alterations in sperm ATP content, motility and viability in bovine semen samples immediately after thawing and following post-thaw incubation at physiological temperature. Two different cryopreservation methods were compared. Ejaculates from ten young bulls were split into two and cryopreserved using conventional procedure with Biladyl (B) extender and with SpermVital (SV) immobilization technology. From each sample, simultaneous analysis of ATP content, motility and viability was performed post-thaw (T0) and after incubation at physiological temperature for three hours (T3). Multivariate correlation analysis showed high correlation at T0 between ATP content and viability (p < 0.05), ATP and total motility (p < 0.05), as well as progressive motility and viability (p < 0.05). However, there was no significant correlation between progressive motility and ATP content at T3, neither for B nor SV semen. We conclude that both preservation method and post-thaw incubation at physiological temperature affect ATP level in bull sperm cells partly independent of motility and viability. The ATP level of bovine spermatozoa post-thaw is therefore implicated to give supplementary information of sperm quality.