PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.