The pharmacokinetic profiles of two iodinated human epidermal growth factors (125I-hEGF51 and 125I-hEGF53) in rats receiving a single intravenous dose have been investigated using three independent bioanalytical techniques. Based on a tetrachloroacetic acid precipitation methodology, hEGF51 and hEGF53 were found to have distribution half-lives of 0.80 +/- 0.2 and 0.80 +/- 0.18 min, and elimination half-lives of 79.8 +/- 24.1 and 77.9 +/- 21.1 min, respectively. Evaluated by immunoprecipitation, distribution half-lives were 0.59 +/- 0.09 and 0.63 +/- 0.15 min, and elimination half-lives were 117.8 +/- 22.9 and 118.7 +/- 38.8 min, respectively. Both precipitation techniques produced similar, parallel plasma concentration-time curves, and there were no significant differences in other calculated kinetic parameters, including clearance and volume of distribution evaluated by either technique. Plasma disposition profiles of both peptides were also confirmed by visualization with SDS-PAGE and autoradiography, and were found to be similar to those generated by tetrachloroacetic acid and immunoprecipitation methods. Thus, three independent methods strongly suggest that both peptides have the same disposition profile, which exhibits a very rapid disappearance rate in the distribution phase followed by a much slower elimination process. These results also indicate that the pharmacokinetic behavior of human epidermal growth factor is not altered by deletion of two amino acids from the carboxyl terminus. In addition, the incubation study suggests that about 23% of the exogenous peptides were associated with red blood cells.