CL (cardiolipin) is a major mitochondrial membrane phospholipid important for the regulation of mitochondrial function. We examined CL de novo biosynthesis and its resynthesis in isolated rat liver hepatocytes prepared 48 h subsequent to two-thirds PHx (partial hepatectomy). The pool size of CL and its de novo biosynthesis from [1,3-(3)H]glycerol were increased 3.3-fold (P<0.05) and 3.1-fold (P<0.05) respectively in hepatocytes prepared from PHx rats compared with sham-operated controls. The reason for the increased CL biosynthesis was a 65% increase (P<0.05) in enzymic activity in PGP-S (phosphatidylglycerolphosphate synthase), a key enzyme in de novo CL biosynthesis. The increase in PGP-S activity was due to a 3-fold increase (P<0.05) of hepatic PGP-S mRNA expression. The increase in de novo CL biosynthesis and pool size corresponded to a 2.3-fold increase (P<0.05) in the amount of [1-14C]linoleic acid incorporated into CL of hepatocytes prepared from PHx rats compared with sham-operated controls, indicating an increase in CL resynthesis. The activity of MLCL-AT (monolysocardiolipin acyltransferase), a rate-limiting enzyme of CL resynthesis, was increased by 43% (P<0.05) in hepatocytes prepared from PHx rats compared with sham-operated controls; this result would explain the increase in [1-14C]linoleic acid incorporation into CL. The increase in MLCL-AT activity was due to an increase in hepatic MLCL-AT protein expression. The results show that CL de novo biosynthesis and its resynthesis are increased during liver regeneration.