The endpoints of the large inverted repeat (IR) of chloroplast DNA in flowering plants differ by small amounts between species. To quantify the extent of this movement and define a possible mechanism for IR expansion, DNA sequences across the IR-large single-copy (IR-LSC) junctions were compared among 13 Nicotiana species and other dicots. In most Nicotiana species the IR terminates just upstream of, or somewhere within, the 5' portion of the rps19 gene. The truncated copy of this gene, rps19', varies in length even between closely related species but is of constant size within a single species. In Nicotiana, six different rps19' structures were found. A phylogenetic tree of Nicotiana species based on restriction site data shows that the IR has both expanded and contracted during the evolution of this genus. Gene conversion is proposed to account for these small and apparently random IR expansions. A large IR expansion of over 12 kb has occurred in Nicotiana acuminata. The new IR-LSC junction in this species lies within intron 1 of the clpP gene. This rearrangement occurred via a double-strand DNA break and recombination between poly (A) tracts in clpP intron 1 and upstream of rps19. Nicotiana acuminata chloroplast DNA contains a "molecular fossil' of the IR-LSC junction that existed prior to this dramatic rearrangement.