The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa, The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(-)), Under greenhouse conditions, Thy(-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(-) rhizobia contained only Thy(+) revertants. In soil, Thy(-) rhizobia were compromised and failed to nodulate alfalfa, Thy(-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY, At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602, The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.