Analogues of E. coli dihydrofolate reductase (DHFR) containing modified amino acids at single, predetermined sites have been prepared. This was accomplished by the use of the DHFR gene containing an engineered nonsense codon (TAG) at the positions corresponding to Val-10 and Asp-27. Misacylated suppressor tRNAs activated with the modified amino acids of interest were employed for the suppression of the nonsense codons in a cell free protein biosynthesizing system, thereby permitting the elaboration of the desired protein analogues. In this fashion, the aspartic acid analogues erythro-carboxyproline, cysteic acid, beta,beta-dimethylaspartic acid, alpha-methylaspartic acid, erythro- and threo-beta-methylaspartic acid, N-methylaspartic acid, and phosphonoalanine were incorporated into one or both of the aformentioned positions. Although a number of these analogues were incorporated only in low yield, a modification of the strategy has suggested how this might be improved significantly. The derived proteins were purified and then characterized by their mobility on polyacrylamide gels in comparison with wild-type DHFR. Representative DHFRs modified at position 10 were also degraded by defined proteolysis with Glu-C endoproteinase; the fragments containing the modified amino acids were shown to have the same chromatographic properties on reverse phase HPLC as authentic synthetic standards. individual analogues were assayed for their abilities to bind to the substrate analogue methotrexate and to convert dihydrofolate to tetrahydrofolate. DHFR analogues containing erythro-and threo-beta-methylaspartic acid and beta,beta-dimethylaspartic acid were all shown to mediate tetrahydrofolate production 74-86% as efficiently as wild-type DHFR under conditions of multiple substrate turnover. Analysis of the rates of tetrahydrofolate production in the presence of NADPH and NADPD at two pH values suggests that this was due to rate-limiting hydride transfer from NADPH bound to DHFR analogues whose active site had been altered structurally.