The estrogen receptor (ER) is a target for endocrine therapy in breast cancer patients. Individual quantification of ER alpha and ER beta expression, rather than total ER levels, might enable better prediction of the response to treatment. We recently developed the tracer 2-F-18-fluoro-6-(6-hydroxynaphthalen-2-yl) pyridin-3-ol (F-18-FHNP) for assessment of ER beta levels with PET. In the current study, we investigated several pharmacokinetic analysis methods to quantify changes in ER beta availability with F-18-FHNP PET. Methods: Male nude rats were subcutaneously inoculated in the shoulder with ER alpha/ER beta-expressing SKOV3 human ovarian cancer cells. Two weeks after tumor inoculation, a dynamic F-18-FHNP PET scan with arterial blood sampling was acquired from rats treated with vehicle or various concentrations of estradiol (nonspecific ER agonist) or genistein (ER beta-selective agonist). Different pharmacokinetic models were applied to quantify ER beta availability in the tumor. Results: Irreversible-uptake compartmental models fitted the kinetics of F-18-FHNP uptake better than reversible models. The irreversible 3-tissue-compartment model, which included both the parent and the metabolite input function, gave results comparable to those of the irreversible 2-tissue-compartment model with only a parent input function, indicating that radioactive metabolites contributed little to the tumor uptake. Patlak graphical analysis gave metabolic rates (K-i, the irreversible uptake rate constant) comparable to compartment modeling. The K-i values correlated well with ER beta expression but not with ER alpha, confirming that K-i is a suitable parameter to quantify ER beta expression. SUVs at 60 min after tracer injection also correlated (r(2) - 0.47; P - 0.04) with ER beta expression. A reduction in F-18-FHNP tumor uptake and Ki values was observed in the presence of estradiol or genistein. Conclusion: F-18-FHNP PET enables assessment of ER beta availability in tumor-bearing rats. The most suitable parameter to quantify ER beta expression is the K-i. However, a simplified static imaging protocol for determining the SUVs can be applied to assess ER beta levels.