P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two ATP-binding cassette efflux transporters that are coexpressed at the human blood-brain barrier (BBB) and blood-retina barrier (BRB). While pharmacological inhibition of P-gp and/or BCRP results in increased brain distribution of dual P-gp/BCRP substrate drugs, such as the tyrosine kinase inhibitor erlotinib, the effect of P-gp and/or BCRP inhibition on the retinal distribution of such drugs has hardly been investigated. In this study, we used positron emission tomography (PET) imaging to assess the effect of transporter inhibition on the distribution of [¹¹C]erlotinib to the human retina and brain. Twenty two healthy volunteers underwent two PET scans after intravenous (i.v.) injection of a microdose (<5 μg) of [¹¹C]erlotinib, a baseline scan, and a second scan either with concurrent i.v. infusion of tariquidar to inhibit P-gp (n= 5) or after oral intake of single ascending doses of erlotinib (300 mg, 650 mg, or 1000 mg,n= 17) to saturate erlotinib transport. In addition, transport of [³H]erlotinib to the retina and brain was assessed in mice by in situ carotid perfusion under various drug transporter inhibition settings. In comparison to the baseline PET scan, coadministration of tariquidar or erlotinib led to a significant decrease of [¹¹C]erlotinib total volume of distribution (V_T) in the human retina by -25 ± 8% (p≤ 0.05) and -41 ± 16% (p≤ 0.001), respectively. In contrast, erlotinib intake led to a significant increase in [¹¹C]erlotinibV_Tin the human brain (+20 ± 16%,p≤ 0.001), while administration of tariquidar did not result in any significant changes. In situ carotid perfusion experiments showed that both P-gp and BCRP significantly limit the distribution of erlotinib to the mouse retina and brain but revealed a similar discordant effect at the mouse BRB and BBB following co-perfusion with tariquidar and erlotinib as in humans. Co-perfusion with prototypical inhibitors of solute carrier transporters did not reveal a significant contribution of organic cation transporters (e.g., OCTs and OCTNs) and organic anion-transporting polypeptides (e.g., OATP2B1) to the retinal and cerebral distribution of erlotinib. In conclusion, we observed a dissimilar effect after P-gp and/or BCRP inhibition on the retinal and cerebral distribution of [¹¹C]erlotinib. The exact mechanism for this discrepancy remains unclear but may be related to the function of an unidentified erlotinib uptake carrier sensitive to tariquidar inhibition at the BRB. Our study highlights the great potential of PET to study drug distribution to the human retina and to assess the functional impact of membrane transporters on ocular drug distribution.