We have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and 'prototype' plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.