A DNA-directed coupled transcription-translation system has been developed in cell-free extracts from the facultative phototroph, Rhodopseudomonas sphaeroides. The in vitro protein-synthesizing system was active when prepared from either chemoheterotrophically or photoheterotrophically grown cells. Optimal activity was dependent upon: use of extracts prepared freshly from early exponential phase cells, the method of cell breakage, and the length of preincubation of the extract (S-30), as well as the concentrations of S-30, template, and cations. The R. sphaeroides cell-free system was compared to one prepared from Escherichia coli. DNA templates tested included R. sphaeroides phage RS1 DNA and E. coli phages T4 and T7 DNA, as well as plasmids RSF1010, pBR322, pSL25 (a pBR322 derivative), and a chimeric plasmid of pSL25 and RSF1010. One RNA template, phage R17, was also employed to test translational fidelity. Transcriptional-translational specificity was observed between R. sphaeroides and E. coli and these observations are discussed in terms of differential gene expression among phylogenetically distinct groups of bacteria.