A method was developed for quantitative analysis of molecular species of ceramide (N-acyl-sphingosine) and dihydroceramide (N-acyl sphinganine) by high performance liquid chromatography (HPLC). Various N-acyl chain-containing ceramides or dihydroceramides were semi-synthesized as standard materials and allowed to react with anthroyl cyanide, a fluorescent reagent. Anthroyl derivatives of ceramide and dihydroceramide containing C16, C18, C20, C22, and C24 saturated N-acyl chain could be completely separated to each molecular species by reversed-phase HPLC equipped with fluorescence detector, although some ceramide molecular species containing monoenoic acyl chain were eluted together with saturated dihydroceramide species. Ceramide molecular species could be quantified using N-heptadecanoyl or N-tricosanoyl sphingosine as an internal standard, and the lower detection limit was below 1 pmol. This method was applied to the analysis of sphingomyelin and free ceramide in U937 cells. The analysis of the ceramide obtained by hydrolysis of sphingomyelin of U937 cells revealed that the ceramide moiety was mainly composed of N-palmitoyl sphingosine, N-nervonoyl sphingosine, and N-lignoceroyl sphingosine, representing 50.0, 27.4, and 6.7% of sphingomyelin, respectively. The total free ceramide and dihydroceramide of U937 cells was determined to be 254 +/- 5 pmol/10(6) cells. Major molecular species of the free ceramide fraction were N-lignoceroyl, N-palmitoyl, and N-nerovonoyl sphingosine, representing 27.6%, 26.6%, and 13.6% of this fraction, respectively. Different distribution of free ceramide molecular species from sphingomyelin species may suggest that selective metabolism of molecular species occurs in the synthesis or degradation of sphingomyelin. These results indicate that the picomole level of molecular species of ceramide and dihydroceramide is successfully determined by fluorescence HPLC and that this newly developed method may be useful to reveal the metabolism and function of ceramide and related compounds in cultured cells.