Ceramide kinase-like (CerkL) is the most recently identified member of the sphingolipid metabolizing enzyme family. This protein is believed to have ceramide kinase (CerK) activity; however, this has not been clarified yet. We generated CerkL-deficient (CerkL(-/-)) mice, measured ceramide (Cer) and ceramide-1-phosphate (C1P) levels in isolated retina, and compared them to levels measured in Cerk(-/-) and WT retinas. We also labeled CerkL(-/-), Cerk(-/-), and WT retinas with P-33 orthophosphate to measure and compare de novo phosphorylation of Cer. Whereas Cerk(-/-) retinas displayed decreased C1P and enhanced Cer, and lacked the capacity to phosphorylate Cer, CerkL(-/-) retinas were indistinguishable from WT retinas with regard to Cer and C1P levels, and in their ability to phosphorylate Cer. Altogether, our results do not support the hypothesis that CerkL, is a second CerK enzyme impacting on Cer levels in the retina. CerkL, if active enzymatically, might use a novel, not yet described, lipid substrate. (c) 2008 Elsevier Inc. All rights reserved.