During oestrus the plasma progesterone concentration can be used to determine the optimal time for mating in the dog. In this study, the optimal time for mating for 26 bitches was determined by measuring progesterone concentrations with three ELISA test kits and a 125I-radioimmunoassay (125I-RIA). Two observers evaluated the ELISA results simultaneously. They graded the results independently and did not know which dog was being tested. For both observers, the optimal time for mating, based on progesterone concentrations measured with the Progesterone Small Rapid ELISA test and the Status-Pro test, differed significantly from that based on progesterone concentrations measured by 125I-RIA. Although the optimal time for mating based on the Ovucheck Premate test did not differ significantly from that based on the 125I-RIA, three out of 14 dogs (21%) for observer one and four out of 14 dogs (29%) for observer two would have been mated on a different day than that determined on the basis of results obtained with the 125I-RIA. The results did not change when the advised period for mating based on the RIA results was extended by two days. It is concluded that the progesterone concentrations measured with the ELISA test kits are too inaccurate for clinical use to determine the optimal time for mating. The only reliable results for determining the optimal mating period are obtained when plasma progesterone concentrations are measured with a RIA.