We describe a selection procedure for construction of very high affinity variants of human growth hormone (hGH) for binding to the extra cellular domain of its receptor (called the hGHbp). Five different libraries of mutated hGH genes (each containing approximately 2 x 10(5) protein variants) were created by randomly mutating four different codons at residues that were shown to be important for receptor binding by structural or functional criteria. Mutated proteins were displayed as single copies from their respective filamentous phagemid particles and sorted in vitro for binding to the immobilized hGHbp. Phagemid particles that bound the immobilized hGHbp were eluted and propagated. After three to seven rounds of binding enrichments, hGH variants were isolated that contained 2 to 4 mutations and exhibited three- to sixfold improvements in binding affinity. Because of the limits of DNA transfection efficiency in creating the library we could not sample thoroughly mutations at more than four codons at once. Nonetheless, the free energy effects for these mutations acted cumulatively. Thus, by combining affinity enhanced mutants from libraries independently sorted we created an hGH variant with 15 substitutions that bound approximately 400-fold more tightly to the hGHbp than wild-type hGH. The affinity enhancements occurred predominantly by slowing the off-rate of the hormone (> 60-fold), and partly through increasing the on-rate (up to 4-fold). Residues that were shown to be important for binding by alanine-scanning were most highly conserved after binding selection, and interestingly many of them could be further improved. Thus, we found it most effective to randomly mutate the residues that were shown to modulate affinity by alanine-scanning, and to combine the selectants from separate libraries that exhibit the highest affinities. The selection procedure and mutagenesis strategy provides a framework for affinity maturation of protein-protein complexes.