The effects of anticoagulant, storage time, storage temperature, and assay method, on laboratory measurements of blood progesterone concentrations of dogs is unclear; these factors have had a dramatic effect on blood progesterone concentrations in other species (particularly cows). In six experiments, we determined the effects of assay technique (chemiluminescence versus radioimmunoassay (RIA)), storage time, and temperature, as well as the use of heparinized plasma versus serum (coagulated blood) on measured progesterone concentrations of bitches. The studies showed that: (a) RIA measured significantly higher serum progesterone concentration (SPC) than chemiluminescence; (b) refrigeration of whole blood during the first 2 h after sample collection significantly decreased measured SPC; (c) progesterone concentration in heparinized plasma was not affected by storage temperature of whole blood for at least 5 h; (d) refrigeration of whole, clotted blood did not affect SPC, provided that samples were held at room temperature for the first 2 h after collection. These findings are of particular importance when blood samples are collected for determination of the initial rise in SPC that is associated with the LH surge in estrous bitches.