A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cmr, Tcr, and Emr determinants that are expressed in E. coli. Only the Emr determinant is expressed in S. sanguis. Both the Cmr and the Tcr of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras into S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345-353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.