Several factors can influence ortholog replacement between closely related species. We evaluated the transcriptional expression and metabolic performance of ortholog substitution complementing a Sinorhizobium meliloti argC mutant with argC from Rhizobiales (Agrobacterium tumefaciens, Rhizobium etli, and Mesorhizobium loti). The argC gene is necessary for the synthesis of arginine, an amino acid that is central to protein and cellular metabolism. Strains were obtained carrying plasmids with argC orthologs expressed under the speB and argC (S. meliloti) and lac (Escherichia coli) promoters. Complementation analysis was assessed by growth, transcriptional activity, enzymatic activity, mRNA levels, specific detection of ArgC proteomic protein, and translational efficiency. The argC orthologs performed differently in each complementation, reflecting the diverse factors influencing gene expression and the ability of the ortholog product to function in a foreign metabolic background. Optimal complementation was directly related to sequence similarity with S. meliloti, and was inversely related to species signature, with M. loti argC showing the poorest performance, followed by R. etli and A. tumefaciens. Different copy numbers of genes and amounts of mRNA and protein were produced, even with genes transcribed from the same promoter, indicating that coding sequences play a role in the transcription and translation processes. These results provide relevant information for further genomic analyses and suggest that orthologous gene substitutions between closely related species are not completely functionally equivalent.