Relaxin isolated from the ovaries of pregnant rats has a mol wt of 6,500. This study determined the size(s) of the relaxin-immunoactive component(s) in the serum of rats during pregnancy. Peripheral sera from rats on days 15, 17, 19, and 21 of pregnancy were filtered through Sephacryl S-200 columns at 37 C and 4 C. Relaxin immunoactivity eluted in three components designated C1, C2, and C3, which had mol wts of approximately 60,000, 13,000 and 6,500, respectively. The distribution of relaxin immunoactivity among C1, C2, and C3 changed with the day of pregnancy. On day 15 of pregnancy essentially all relaxin immunoactivity was associated with C1. By day 17 approximately 20% of the relaxin immunoactivity was in C2 plus C3, and by day 21 the percentage in each of these two components increased to about 25%. C1 appears to possess relaxin bioactivity since the frequency of intrauterine pressure cycles in nonpregnant rats declined during cross-circulation with day 15 pregnant rats. C1 may not be attributable to the binding of 6,500 mol wt relaxin to a binding protein(s) since relaxin immunoactivity remained associated with a component(s) having a mol wt greater than 28,000 after incubation and gel filtration of day 15 serum in the presence of 2.0 M potassium thiocyanate at 37 C. Moreover, the levels of C1 as well as those of C2 and C3 were markedly increased in the peripheral serum of day-21 rats within 15 min of LH administration. C3 may be the 6,500-mol wt form of relaxin and C2 may be an intermediate in the processing of the 20,000-mol wt rat relaxin precursor to the 6,500-mol wt form of relaxin. The physiological significance, if any, of the multiplicity of relaxin-immunoactive components is unknown.