Long-chain unsaturated fatty acids inhibit several cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes involved in drug metabolism, including CYP2C8, CYP2C9, UGT1A9, UGT2B4, and UGT2B7. Bovine serum albumin (BSA) enhances these cytochrome P450 and UGT activities by sequestering fatty acids that are released from membranes, especially with human liver microsomes (HLM) as the enzyme source. Here, we report the effects of BSA on CYP1A2-catalyzed phenacetin (PHEN) O-deethylation and lidocaine (LID) N-deethylation using HLM and Escherichia coli-expressed recombinant human CYP1A2 (rCYP1A2) as the enzyme sources. BSA (2% w/v) reduced (p < 0.05) the K(m) values of the high-affinity components of human liver microsomal PHEN and LID deethylation by approximately 70%, without affecting V(max). The K(m) (or S(50)) values for PHEN and LID deethylation by rCYP1A2 were reduced to a similar extent. A fatty acid mixture, comprising 3 μM concentrations each of oleic acid and linoleic acid plus 1.5 μM arachidonic acid, doubled the K(m) value for PHEN O-deethylation by rCYP1A2. Inhibition was reversed by the addition of BSA. K(i) values for the individual fatty acids ranged from 4.7 to 16.7 μM. Single-point in vitro-in vivo extrapolation (IV-IVE) based on the human liver microsomal kinetic parameters obtained in the presence, but not absence, of BSA predicted in vivo hepatic clearances of PHEN O-deethylation and LID N-deethylation that were comparable to values reported in humans, although in vivo intrinsic clearances were underpredicted. Prediction of the in vivo clearances of the CYP1A2 substrates observed here represents an improvement on other experimental systems used for IV-IVE.