The present study reports liver carnitine biosynthesis and concentrations in non-pregnant (dry), pregnant and lactating rabbits (n = 17). To determine carnitine biosynthesis in liver tissues; we purified gamma-butyrobetaine hydroxylase, a key enzyme in carnitine biosynthesis, and measured its activity. An isocratic high-performance liquid chromatography (HPLC) method was used to determine L-carnitine levels in liver samples. When compared with dry period, during pregnancy, free and acid-soluble total carnitine concentrations did not change significantly (p>0.05). In contrast to free and acid-soluble total carnitine, short chain acylcarnitine concentration increased significantly in the second period of pregnancy, compared with non-pregnant rabbits. Highest concentrations of free, short-chain acyl- and acid-soluble total carnitine were measured at the 4th day of lactation (487 +/- 29; 392 +/- 26 and 879 +/- 38 nmol/g wet weight respectively). gamma-Butyrobetaine hydroxylase activity increased with the duration of pregnancy and in late pregnancy it increased significantly compared to previous periods. Highest activity was measured in early lactation period (361 +/- 28 pmol carnitine/min/mg protein). We concluded that, biosynthesis of carnitine in liver increases gradually in late pregnancy and early lactation in rabbit, and this may be regulated by hormonal changes during these periods.