We assessed the serum concentration of γ‐carboxylated osteocalcin (OC), total OC, and full‐length OC in a clinical setting of 37 patients on continuous warfarin treatment (international normalized ratio 2.0–3.8). A comparison was done with the results from 30 untreated age‐matched controls. Four monoclonal antibodies, previously generated and characterized as to their ability to recognize different human OC forms and fragments, were used in three two‐site immunofluorometric assays. The warfarin‐treated patients had significantly lower levels of carboxylated OC 4.9 ± 3.8 (± 1 SD) ng/ml compared with the controls 13.1 ± 9.7 (p < 0.0001). There was no difference in the levels of total OC or full‐length OC between the two groups of patients. A strong correlation was found between the serum concentration of carboxylated OC and total OC, both for the warfarin‐treated patients (r = 0.98) and for the controls (r = 0.99). There was a distinct cut‐off level at 0.80, in the quotient carboxylated OC/total OC, at which all warfarin‐treated patients fell below and all controls above this level. Hence, the concentration or ratio of serum γ‐carboxylated OC in clinical settings such as warfarin‐treated patients could be measured using two‐site immunoassays.