Canine relaxin (cRlx) was synthesized by a combination of solid-phase methods and sequential site-directed disulfide bond formation. Proof that the intended molecule had been synthesized was obtained by analytical HPLC of the intact and reduced molecule, by amino acid and sequence analysis, and by receptor binding and in vivo mouse interpubic ligament bioassays. Antisera to synthetic cRlx were raised in six male rabbits; these cross-reacted with relaxins of other species, but not with insulin, LH, FSH, hCG, or prolactin (PRL). Three of the antisera neutralized relaxin-induced interpubic ligament formation in estrogen-primed mice in vivo. A new homologous cRlx RIA was developed through the use of rabbit antiserum 79888, synthetic cRlx for standards and 125l-labeled trace, and a goat anti-rabbit lgG-polyethylene glycol precipitant. The new RIA can be completed in 26 h and has a sensitivity of 0.195-0.39 ng cRlx/tube. Intra- and interassay coefficients of variation were 3% and 12.5%. During pregnancy in bitches, serum cRlx rose to about 10 micrograms/ml. Immunoactive cRlx was also detected in serum, colostrum, and milk of lactating bitches, but not in large volumes (100-300 microliters) of serum of pseudopregnant or estrous bitches. Immunoreactive cRlx was also found in seminal plasma, but not in serum, of male dogs. The new homologous cRlx RIA is simple, rapid, sensitive, and specific, and will be used in future studies of canine relaxin physiology.