Various studies indicate that cytodifferentiation of Clara cells and development of pulmonary cytochrome P450 (CYP) monooxygenases occur postnatally. The timing of these events is species-specific. Neonatal mice are more susceptible than adult mice are to Clara cell injury by naphthalene, but little is known about the postnatal development of Clara cells and CYP in mice. This study was designed to determine the developmental pattern of Clara cell differentiation and CYP expression in mice. Lungs from mice aged 16 days gestation to 63 days postnatal (DPN) were studied. Clara cell secretory protein (CC10) expression in nonciliated cells was detected earlier in proximal airways than in distal airways, but reached adult levels at 14 DPN in all airway levels. Cilia-associated tubulin expression closely followed the onset of CC10 expression, as did expression of CYP reductase. CYP2B protein expression appeared and differentiated earlier in bronchi than in bronchioles and reached adult levels at 14 and 28 DPN, respectively. CYP2F2 expression appeared earlier in proximal airways, but did not reach adult levels of expression until after 28 DPN. CYP activity, measured by naphthalene metabolism, increased with age and corresponded to CYP2F2 protein expression. We conclude that in the mouse, (1) Clara cell maturation is a postnatal event, (2) Clara cell differentiation is complete at the same age in proximal and distal airways, (3) CYP reductase protein expression occurs at the same time as CC10 expression, but CYP2B and CYP2F2 lag behind, and (4) stereoselective naphthalene monooxygenase activity corresponds with CYP2F2 protein expression.