The regulatory role of protein kinase C (PKC) δ isoform in the stimulation of phospholipase D (PLD) by sphingosine-1-phosphate (SPP) in a human-airway epithelial cell line (CFNPE9o−) was revealed by using antisense oligodeoxynucleotide to PKCδ, in combination with the specific inhibitor rottlerin. Cell treatment with antisense oligodeoxynucleotide, but not with sense oligodeoxynucleotide, completely eliminated PKCδ expression and resulted in the strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKCα, β, δ, ∊ and ζ isoforms expressed in these cells, only PKCδ was activated on cell stimulation with SPP, as indicated by translocation into the membrane fraction. Furthermore, pertussis toxin and genistein eliminated both PKCδ translocation and PLD activation. In particular, a significant reduction in phosphatidylbutanol formation by SPP was observed in the presence of 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP1), an inhibitor of Src tyrosine kinase. Furthermore, the activity of Src kinase was slightly increased by SPP and inhibited by PP1. However, the level of PKCδ tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCδ. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas the PLD1 isoform alone was threonine-phosphorylated in SPP-treated cells. PLD1 threonine phosphorylation was strongly inhibited by rottlerin, by anti-PKCδ oligodeoxynucleotide and by PP1. In conclusion, in CFNPE9o− cells, SPP interacts with a membrane receptor linked to a Gi type of G-protein, leading to activation of PLD, probably the PLD1 isoform, by a signalling pathway involving Src and PKCδ.