The present study was conducted to develop and validate a specific radioimmunoassay system for measurement of bovine chymosin B (bChyB) concentrations in plasma samples. Bovine ChyB was used for immunization of rabbits and as standard and tracer. Chymosin B concentrations were measured in plasma samples from two groups of calves (Group 1: calves sampled from birth to 24 hours; Group 2: calves sampled from Day 1 to 21 after birth) and from one cow during the peri-partum period. Detection limit of the assay was 9.0 ng/ml. Recovery was higher than 89.3%. Repeatability and reproducibility ranged from 1.52% to 5.23% and from 1.52% to 12.57% respectively. No cross-reaction was found with pepsinogen A from bovine, porcine or human origins. In Group 1, bChyB concentrations increased from 47.3 ± 45.1 ng/ml (5 min after birth) to 325.5 ± 161.2 ng/ml (12 hours after birth), then no significant change was observed till 24 hours after birth (293.0 ± 161.5 ng/ml). In Group 2, concentrations decreased from Day 1 (455.3 ± 191.1 ng/ml) to Day 21 (117.9 ± 85.1 ng/ml). In adult cow, mean concentration was 136.0 ± 32.3 ng/ml. In conclusion, bChyB is able to cross the stomach basal membrane and to reach the blood circulation at detectable levels in both young calves and adult cows.