A monoclonal antibody (MAb) to phenobarbital-induced rat cytochrome P450b was used to study the interaction of the substrate benzphetamine (Bz) with cytochromes P450 in liver microsomes. Binding of Bz to liver microsomes from phenobarbital-treated rats was monitored by the substrate-induced type I spectral change. The MAb maximally inhibited this spectral change by 49%, providing a probe to distinguish MAb-specific P450b from other Bz-binding P450s. Thermodynamic parameters of the interaction were determined in the absence and presence of MAb. The MAb did not influence the spin-state equilibrium of substrate-free P450b, but it increased the low spin content of substrate-bound P450b. The MAb also decreased the affinity of both high and low spin P450b for Bz. The temperature dependence of the Bz-binding interactions revealed a transition near 20 degrees C. Fluorescence polarization measurements of the membrane probe 1,6-diphenyl-1,3,5-hexatriene also revealed a transition at this temperature. The MAb comparably inhibited Bz binding to high spin P450b in the low and high temperature regions, whereas MAb inhibition of Bz binding to low spin P450b was greater in the low temperature region than in the high temperature region. These results indicate temperature-dependent changes in membrane structure that modulate both Bz binding to P450b and MAb-P450b-Bz interactions. These results also demonstrate the utility of MAbs for evaluating P450-substrate binding microequilibria of MAb-specific P450s in the presence of other P450s while in the natural membrane environment of the endoplasmic reticulum.