Cholesterol 7α-hydroxylase is a rate-limiting enzyme in bile acid synthesis, a major pathway for cholesterol catabolism. It plays a crucial role in postnatal development and survival. In an adult liver, its activity and messenger RNA (mRNA) are heterogeneously distributed with concentration in the pericentral area. We defined how the pattern of cholesterol 7α-hydroxylase mRNA evolves during rat liver development, correlated this with its total liver mRNA levels, and determined when its heterogeneous pattern of expression is established. Cholesterol 7α-hydroxylase mRNA was undetectable in 18-day-old fetal livers by Northern blot. It was increased markedly in newborns with a homogeneous liver lobular distribution as determined by in situ hybridization. At postnatal day four, mRNA levels were markedly decreased with concomitant appearance of a lobular gradient: mRNA was detected only in a few hepatocytes located around efferent venules. At 22 days, the time of highest mRNA expression, a marked extension of the gradient towards the periportal area was observed, indicating that the increase in total liver cholesterol 7α-hydroxylase mRNA level was a result of recruitment of hepatocytes upstream from the central vein area. By 28 days, the adult pattern was observed. Thus, expression of cholesterol 7α-hydroxylase mRNA is tightly regulated during rat liver development, both temporally and spatially supporting its critical role in normal postnatal development.