Ceramide is involved in the regulation of many cellular processes including cell proliferation and apoptosis, which are accompanied respectively with a decrease and an increase in the activity of the Na(+)/K(+) ATPase. These antagonistic effects may be time-dependent and due to different signaling pathways requiring different time intervals to be activated. While we showed previously a ceramide-induced inhibition of the ATPase in HepG2 cells during the first hour, we study here the effect of ceramide thereafter. Ceramide stimulated the Na(+)/K(+) ATPase between 1 and 4h with a peak at 2h. This stimulation was maintained in the simultaneous presence of an inhibitor of ceramidase (CAY 10466) but disappeared when ceramide kinase was inhibited, suggesting a role of ceramide-1-phosphate (cer-1-P) in the observed effect. Exogenous cer-1-P caused a similar stimulation of the ATPase which was not affected by an inhibition of JNK but changed into a decrease in presence of PDTC, a specific inhibitor of NF-κB, and disappeared when NF-κB and JNK were inhibited simultaneously. It was concluded that cer-1-P activates both JNK and NF-κB. While JNK exerts an inhibitory effect on the ATPase, NF-κB increases its activity and abrogates the stimulatory effect of the sphingolipid on JNK leading thus to an additional increase in the ATPase activity.