Plasma sphingosine-1-phosphate (S1P) has been suggested to mainly originate from erythrocytes; however, within the erythrocyte, how sphingosine (SPH) generation--the precursor to S1P--is controlled is unknown. SPH is only generated from the hydrolysis of ceramides via ceramidases. Five human ceramidases have been identified: 1 acid, 1 neutral, and 3 alkaline ceramidases (ACER1, ACER2, and ACER3). Here, we demonstrate that only alkaline ceramidase activity is expressed in erythrocytes and that it is instrumental for SPH generation. Erythrocytes have alkaline but not acid or neutral ceramidase activity on D-e-C(18:1)-ceramide, a common substrate of ceramidases. Not only alkaline ceramidase activity but also the generation of SPH and S1P are increased during erythroid differentiation in K562 erythroleukemic cells. Such SPH and S1P increases were inhibited by the alkaline ceramidase inhibitor D-e-MAPP, suggesting that alkaline ceramidases have a role in the generation of SPH and S1P in erythroid cells. Alkaline ceramidase activity is highly expressed in mouse erythrocytes, and intravenous administration of D-e-MAPP decreased both SPH and S1P in erythrocytes and plasma. Collectively, these results suggest that alkaline ceramidase activity is important for the generation of SPH, the S1P precursor in erythrocytes.