The wild-type (WT) Cpr(lox/lox) (cytochrome P(450) oxidoreductase, Cpr) mouse is an ideal model to assess the contribution of P(450) enzymes to the metabolic activation and disposition of environmental xenobiotics. In the present study, we examined the effect of in utero exposure to benzo(a)pyrene [B(a)P] aerosol on Sp4 and N-methyl-D-aspartate (NMDA)-dependent systems as well as a resulting behavioral phenotype (object discrimination) in Cpr offspring. Results from in utero exposure of WT Cpr(lox/lox) mice were compared with in utero exposed brain-Cpr-null offspring mice. Null mice were used as they do not express brain cytochrome P(450)1B1-associated NADPH oxidoreductase (CYP1B1-associated NADPH oxidoreductase), thus reducing their capacity to produce neural B(a)P metabolites. Subsequent to in utero (E14-E17) exposure to B(a)P (100 μg/m(3)), Cpr(lox/lox) offspring exhibited: (1) elevated B(a)P metabolite and F(2)-isoprostane neocortical tissue burdens, (2) elevated concentrations of cortical glutamate, (3) premature developmental expression of Sp4, (4) decreased subunit ratios of NR2B:NR2A, and (5) deficits in a novelty discrimination phenotype monitored to in utero exposed brain-Cpr-null offspring. Collectively, these findings suggest that in situ generation of metabolites by CYP1B1-associated NADPH oxidoreductase promotes negative effects on NMDA-mediated signaling processes during the period when synapses are first forming as well as effects on a subsequent behavioral phenotype.