Abstract Phospholipase D (PLD) is distributed widely in mammalian tissues where it is believed to play an important role in the regulation of cell functions and cell fate by a variety of extracellular signals. In this study, we used primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells, grown to confluence on a permeable support, to investigate the possible involvement of PLD in the mechanism of action of hormones that regulate Ca 2+ reabsorption. RT-PCR revealed the presence of transcripts of PLD1b and PLD2, but not PLD1a, in these cultures. Moreover, the expression of substantial amounts of PLD1 protein was demonstrated by Western blotting. To measure PLD activity, cells were labelled with [ 3H]myristic acid after which the PLD-catalysed formation of radiolabelled phosphatidylethanol ([ 3H]PtdEth) was measured in the presence of 1% (v/v) ethanol. Deamino-Cys, D-Arg 8-vasopressin (dDAVP) and N 6-cyclopentyladenosine (CPA), two potent stimulators of Ca 2+ transport across these monolayers, stimulated PLD activity as was indicated by a marked increase in [ 3H]PtdEth. Similarly, ATP, a potent inhibitor of dDAVP- and CPA-stimulated Ca 2+ transport, increased the formation of [ 3H]PtdEth. PLD activity was furthermore increased by 8Br-cAMP and following acute (30 min) stimulation of protein kinase C (PKC) with a phorbol ester (PMA). Chronic PMA treatment (120 h) to downregulate phorbol ester-sensitive PKC isoforms did not affect PLD activation by dDAVP, CPA and 8Br-cAMP, while markedly decreasing the effect of ATP and abolishing the effect of PMA. The PKC inhibitor chelerythrine significantly reduced PLD activation by dDAVP, CPA and 8Br-cAMP, without changing the effect of ATP. The inhibitor only partially reduced the effect of PMA. This study shows that Ca 2+ transporting cells of CNT and CCD contain a regulated PLD activity. The physiological relevance of this activity, which is not involved in Ca 2+ reabsorption, remains to be established.