Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-ocytl β-d-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240–260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent K _m of 146 μM and a V _max of 11.1 nmol/min/mg protein for stearoyl-CoA. The K _m and V _max values toward sphingosine were 171 μM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the K _m and V _max values were 144 μM and 8.5 nmol/min/mg protein, respectively.