Alterations in hemoglobin oxygen affinity can be detected by exposing blood to different PO2 and recording oxygen saturation, a method termed tonometry. It is the gold standard to measure the PO2 associated with 50 % oxygen saturation, the index used to quantify oxygen affinity (P50Tono). P50Tono is used in the evaluation of patients with erythrocytosis suspected to have hemoglobin with abnormal oxygen affinity. Since tonometry is labor intensive and not generally available, we investigated whether accurate estimates of P50 could also be obtained by venous blood gas analysis, co-oximetry, and standard equations (P50Ven). In 50 patients referred for evaluation of erythrocytosis, pH, PO2, and oxygen saturation were measured in venous blood to estimate P50Ven; P50Tono was measured for comparison. Agreement among P50Ven and P50Tono was evaluated (Bland-Altman analysis). Mean P50Tono was 25.8 (range 17.4-34.1) mmHg. The mean difference (bias) of P50Tono-P50Ven was 0.5 mmHg; limits of agreement (95 % confidence limits) were -5.2 to +6.1 mmHg. The sensitivity and specificity of P50Ven to identify the 25 patients with P50Tono outside the normal range of 22.9-26.8 mmHg were 5 and 77 %, respectively. We conclude that estimates of P50 based on venous blood gas analysis and standard equations have a low bias compared to tonometry. However, the precision of P50Ven is not sufficiently high to replace P50Tono in the evaluation of individual patients with suspected disturbances of hemoglobin oxygen affinity.