Rat testis known to contain all of the enzymes required for carnitine biosynthesis also contains high concentration of calmodulin, a protein which may or may not contain trimethyllysine, the major substrate in carnitine biosynthesis. The purpose of this study was to investigate the levels of carnitine and the state of calmodulin N-methylation in rat testes, and to discuss the possibility of the involvement of calmodulin incarnitine biosynthesis. Nonesterified carnitine , acid soluble acyl carnitine, and acid insoluble acyl carnitine of ra tests were 273 nmole, 62nmole, and 4 nmole/g tissue, respectively. Total carnitine level was 339 nmole/g testes tissue. Calmodulin purified from rat tests was assayed for methylation potential using N-methyltransferase from the rat testes. Rat testes calmodulin showed no 3H-methyl incorporation indicating that the calmodulin was trimethylated already by endogenous calmodulin N-methyltransferase. Amino acid composition analysis revealed that the rat testes calmodulin containd one mole of trimethyllysine per mole of calmodulin. These data suggest that testes calmodulin could provide the trimethyllysine needed for the synthesis of carnitine in the rat tests.