A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14(A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104(G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human alpha-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer, pH 7.0, at 5 degrees C, and the structural flexibility of alpha-apohemoglobin, in the absence and presence of human beta-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (EQ) and Förster distances (R0Q) were analyzed by least-squares to determine the goodness of fit (chi R2) for the assumed distribution parameters: average distance r and half-width hw. Data for alpha-apohemoglobin in the absence and presence of beta h chains yielded values for r of 18 and 22 A and hw of 20 and 8.5 A, respectively. Although the increase in r for alpha-apohemoglobin in the presence of beta h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the alpha-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.