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Shortened estrous cycles, embryonic death, and abortion might be associated with insufficient secretion of progesterone by the canine corpora lutea. The aim of this study was to investigate the concentration of progesterone, prolactin, and relaxin in hypoluteoid and control bitches during pregnancy and the nonpregnant cycle. Moreover, canine antibodies against progesterone were analyzed because of a possible connection between embryonic loss associated with hormone changes related to a hormone antibody response. An enzyme-linked immunosorbent assay (ELISA) was developed and optimized for these purposes. Serum samples from 20 short-cycling and 18 control bitches were analyzed. Animals were assigned to pregnant and nonpregnant groups after ultrasound examination. The results show that the nonpregnant, short-cycling bitches had significantly lower progesterone concentrations than the animals of the control group. In German Shepherd dogs, prolactin concentrations were significantly higher in the hypoluteoid pregnant bitches compared with the control group. Relaxin concentrations did not differ significantly between the pregnant hypoluteoid and pregnant control group at any time of measurement. Moreover, increased levels of IgE antibodies against progesterone were found in the serum of six bitches (five short-cycling bitches, one control animal) out of a total of 38 animals. The results indicated a specific binding of the antibodies to progesterone, but a cross-reaction with estradiol and testosterone might occur. The presented data verify the existence of antibodies against progesterone and suggest that these antibodies might play a role in some cases of unexplained pregnancy failure and shortened cycle length.