Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca(2+) suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca(2+)-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca(2+)-independent manner.