The cell density dependence of stationary-phase survival of Rhizobium leguminosarum has been investigated. Following starvation by exhaustion of carbon or nitrogen, but not of phosphorus, the survival of cultures was dependent on the cell density at entry into stationary phase. High-density cultures survived with little or no loss of viability over a 20-day period in stationary phase. In contrast, low-density cultures lost viability rapidly but consisted of a heterogeneous population, a small fraction of which successfully adapted and eventually formed a stable, surviving population. The threshold density above which the cultures survived successfully in stationary phase was dependent on the growth conditions and the strain used. We took advantage of the fact that R. leguminosarum survives poorly following starvation by resuspension in carbon-free medium to demonstrate that cell density-dependent survival was mediated by a component accumulating in the growth medium. The effects of this medium component on survival in resuspension assays could be mimicked by an N-acyl homoserine lactone, N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone, previously demonstrated to have a role in controlling cell density-dependent phenomena in R. leguminosarum. The Sym plasmids pRP2JI and pRL1JI were found to be essential for the production of the extracellular factor, which could also be made in Escherichia coli carrying the cosmid clone pIJ1086 containing a specific region of pRL1JI.