The purpose of the study was to determine if the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, are substrates for the efflux transporter ABCG2, and to investigate the relevance of the ABCG2 421C>A (Q141K) polymorphism to the pharmacokinetics of gefitinib. Gefitinib and erlotinib transport in vitro was studied using HEK293 cells transfected with wild-type ABCG2 or a Q141K clone. Gefitinib pharmacokinetics was determined in 27 cancer patients. was. ABCG2 421C>A and ABCB1 3435C>T genotypes were determined using direct sequencing. Cells expressing wild-type ABCG2 exhibited lower intracellular accumulation of gefitinib and erlotinib at concentrations of 0.1 and 1 microM, and higher efflux at 1 microM than cells lacking ABCG2 (p < 0.05); no significant difference in cellular efflux and accumulation was observed in the variant cell line at lower concentrations nor in the three cell lines at 10 microM. In the presence of the ABCG2 inhibitor fumitremorgin C, cellular accumulation of gefitinib and erlotinib 1 microM was increased in wild-type (p < 0.05), but not in variant or null cells. Gefitinib accumulation during 28 days of treatment (C(ss,min)/C(1,min)) was higher in patients heterozygous at the ABCG2 421C>A locus than those with a wild-type genotype (median, 5.07 vs. 3.60, p = 0.004). No significant associations were observed between the ABCB1 3435C>T genotype and gefitinib pharmacokinetics. In conclusion, gefitinib and erlotinib are ABCG2 substrates, while they inhibit ABCG2 at higher concentrations. A functional variant of ABCG2 is associated with greater gefitinib accumulation at steady-state and may be relevant to toxicity and antitumor activity of EGFR TKIs.