A series of 120-600 kilobase deletions with defined endpoints were made in the 1600-kilobase Rhizobium meliloti megaplasmid pRmeSU47b, by homologous recombination between the IS50 elements of transposon insertions. Utilizing IS 50-mediated homologous recombination we also made defined reductions in deletion size and combined adjacent deletions. Deletion structure was confirmed by phage transduction and Southern hybridization analysis. Collectively these deletions span 1400 kilobases of pRmeSU47b, indicating that the majority of the plasmid is not essential for cell viability. This was further confirmed by the construction of a strain SU47 derivative which carries only 450 kilobases of the pRmeSU47b megaplasmid. Examination of the deletion mutants for phenotype revealed novel loci required for dulcitol, melibiose, raffinose, beta-hydroxybutyrate, acetoacetate, protocatechuate and quinate utilization. Previously unidentified loci required for effective root nodule development and exopolysaccharide synthesis were also found. Various deletion mutants were deficient in dicarboxylate transport, lactose utilization, and thiamine and exopolysaccharide biosynthesis, as predicted from earlier studies of this megaplasmid.