The evolution and recombination of chloroplast genome structure in the fern genus Osmunda were studied by comparative restriction site mapping and filter hybridization of chloroplast DNAs (cpDNAs) from three species -O. cinnamomea, O. cIaytoniana and O. regalis. The three 144 kb circular genomes were found to be colinear in organization, indicating that no major inversions or transpositions had occurred during the approximately 70 million years since their radiation from a common ancestor. Although overall size and sequence arrangement are highly conserved in the three genomes, they differ by an extensive series of small deletions and insertions, ranging in size from 50 bp to 350 bp and scattered more or less at random throughout the circular chromosomes. All three chloroplast genomes contain a large inverted repeat of approximately 10 kb in size. However, hybridizations using cloned fragments from the O. cinnamornea and O. regalis genomes revealed the absence of any dispersed repeats in at least 50% of the genome. Analysis with restriction enzymes that fail to cleave the 10 kb inverted repeat indicated that each of the three fern chloroplast genomes exists as an equimolar population of two isomeric circles differing only in the relative orientation of their two single copy regions. These two inversion isomers are inferred to result from high frequency intramolecular recombination between paired inverted repeat segments. In all aspects of their general organization, recombinational heterogeneity, and extent of structural rearrangement and length mutation, these fern chloroplast genomes resemble very closely the chloroplast genomes of most angiosperms.