The effects of a single course of antenatal betamethasone on S100B protein concentration were investigated in Fisher 344 rats. On day 20 of gestation, pregnant rats were injected twice 8 h apart with either (1) 170 microg kg(-1) body weight betamethasone ("clinically-equivalent dose", equivalent to 12 mg twice, 24 h apart in humans), (2) half of this dose (equivalent to 6 mg) or (3) vehicle. We report reference values for S100B protein in the serum and different brain regions in both genders at 1, 2, and 21 days after birth. Interestingly, S100B concentration showed a time-dependent and brain region-specific pattern of expression. At P1, S100B was higher in the serum of males compared to females. In addition, we show that both doses of betamethasone decreased S100B concentration in the serum of males at P1, whereas in the hippocampus, it was reduced by the clinically-equivalent dose only. This suggests that lowering the dose of antenatal betamethasone may be less detrimental for brain maturation and therefore we reiterate the need for clinical trials with a low dose regimen.