In several positron-emission tomography studies of human subjects, analyses of data from the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor radioligand, [(11)C]WAY-100635 ¿[carbonyl-(11)C]N-(2-(4-methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N -(2 -pyridyl)cyclohexanecarboxamide¿ have shown a discrepancy between the outcome measure k(3)/k(4) (binding potential normalized to cerebellum) as estimated by the simplified reference region method and results obtained by conventional kinetic modeling with an arterial input function. The reference region method has yielded results that are lower than the conventional approach, with the relative underestimation appearing to be an increasing function of k(3)/k(4). We performed simulations on idealized data to identify the source of the discrepancy. Both the simplified reference tissue model (SRTM) and the original full reference tissue model (FRTM) were tested to determine (a) if the error in estimated k(3)/k(4) is dependent on the blood flow in the region of interest relative to the blood flow in the region of reference (R(1)) and on the receptor density in the region of interest (true k(3)/k(4)), and (b) which violation of the reference model assumptions were responsible for this effect. FRTM returned parameter estimates that were independent and accurate if the reference region was constructed precisely as a one-tissue compartment model. SRTM overestimated k(3)/k(4) when the reference region was constructed as a one-tissue compartment model and underestimated k(3)/k(4) when the reference region was constructed as a two-tissue compartment model (which is the case for [(11)C]WAY-100635). In both cases, the magnitude of the error in k(3)/k(4) returned by SRTM was dependent on true R(1) and true k(3)/k(4). In conclusion, the SRTM is associated with a bias in the derivation of k(3)/k(4) that is not a simple scaling factor. This magnitude of these errors should be carefully evaluated for each new radioligand.