Experiments with polyacrylamide gel electrophoresis at steady state conditions, indicated that testosterone, 5 alpha-dihydrotestosterone, 3 alpha-androstanediol and 3-beta-androstanediol bound only to albumin in rat serum. The extent of binding of several C19-steroids to serum proteins was measured in rat blood utilizing equilibrium dialysis. The values for the adult male rat were as follows (mean +/- SEM): testosterone 78.3 +/- 0.4% 5 alpha-dihydrotestosterone 81.8 +/- 0.4%, 3 alpha-androstanediol 95.5 +/- 0.1%; 3 beta-androstanediol 95.2 +/- 0.1%. The values for the latter two are significantly higher than those for the first two which in turn differ significantly from each other. The values for adult female and castrated male serum samples were not significantly different from those of the normal male. The results obtained for the extent of binding of C19-steroids in a 5-g% albumin solution (which is similar to male rat serum albumin concentration) did not differ significantly from values for serum indicating that the binding in the latter is due primarily to albumin. The KA's for C19-steroid binding to albumin were: testosterone 2.22 X 10(4)l/mol; 5 alpha-dihydrotestosterone 3.46 X 10(4)l/mol; 3 alpha-androstanediol 1.36 X 10(5)l/Mol and 3 beta-androstanediol 1.20 XC 10(5)l/mol. When correlated to our previously reported metabolic clearance rats (MCR) it was concluded that binding to serum proteins does not account for the significant difference between the MCR for testosterone and 5 alpha-dihydrotestosterone but may account for the lower MCR's for the 3 alpha- and 3 beta-androstanediols.