Background We investigated the feasibility of left ventricular (LV) and right ventricular (RV) volume and function estimation using a first-pass gated 15O-water PET. This prospective study included 19 patients addressed for myocardial perfusion reserve assessment using 15O-water PET. PET data were acquired at rest and after regadenoson stress, and gated first-pass images were reconstructed over the time range corresponding to tracer first-pass through the cardiac cavities and post-processed using TomPool software; LV and RV were segmented using a semi-automated 4D immersion algorithm. LV volumes were computed using a count-based model and a fixed threshold at 30% of the maximal activity. RV volumes were computed using a geometrical model and an adjustable threshold that was set so as to fit LV and RV stroke volumes. Ejection curves were fitted using a deformable reference curve model. LV results were compared to those obtained using 99mTc-sestamibi gated myocardial SPECT in terms of end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and ejection fraction (EF). Results There was an excellent concordance between rest and stress PET in terms of EDV and ESV (Lin’s coefficient ~ 0.85–0.90), SV (~ 0.80), and EF (~ 0.75) for both ventricles. Correlation with myocardial SPECT was high for LV EDV (Pearson’s R = 0.89, p < 0.001) and ESV (R = 0.87, p < 0.001) and satisfying for LV SV (R = 0.67, p < 0.001) and EF (R = 0.67, p < 0.001). Minimal LV ESV overestimation (+ 4 mL, p = 0.03) and EF underestimation (− 4%, p = 0.01) were observed using PET. Conclusions Biventricular volume and function assessment are achievable using the first-pass PET, and LV parameters correlate well with those derived from gated myocardial SPECT.