The effect of ascorbate (AsA) on the synthesis of carnitine from gamma-butyrobetaine (BB) in primary cultured guinea pig hepatocytes was investigated. The hepatocyte monolayers preloaded with AsA were incubated for 4 h in medium with various concentrations of BB as the precursor of carnitine. The accumulation of carnitine reached a maximum when the cells were incubated with 0.05-1.0 mM BB and significantly decreased with excess BB (5 mM). In contrast, increasing concentrations of AsA supplemented to medium led to an increase in carnitine content, but AsA and total AsA contents in cells decreased by BB supplementation. Regarding the enhancement of hydroxylation of BB in the hepatocytes, AsA was the most effective among such other reducing agents as glutathione and dithiothreitol. Although erythorbate (ErA) also stimulated the hydroxylation of BB, carnitine content in cells preloaded with ErA was only 60% of that with AsA. These results suggest that AsA is specifically required for the hydroxylation of BB. Furthermore, AsA can regulate carnitine synthesis in the primary cultured guinea pig hepatocytes.