An in vitro transcription-translation system was developed using cell-free extracts from the symbiotic nitrogen-fixing bacterium Rhizobium meliloti strain 41. Conditions for preparation of the 30,000 X g supernatant extract and for measurement of protein-synthesizing activity were determined and compared to the activity of an Escherichia coli cell-free system. Genes expressed in the free-living or in the symbiotic state were studied. The product of a recA-like gene (41-kDa protein) was synthesized both in R. meliloti and E. coli extracts, although less efficiently in the heterologous system. In agreement with earlier results obtained in E. coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 X 10(3)-base DNA region carrying genes for nodulation (nod). However, differences in the transcription-translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R. meliloti and E. coli cell-free extracts, and the possible explanations of these findings are discussed.