The chloroplast genomes of higher plants encode several tRNA genes that contain highly conserved type II introns. Using primers specific to conserved 5' and 3' regions within the introns of the genes trnA (tRNA-ala) and trnI (tRNA-ile) we have PCR amplified parts of these introns from 36 plant species representing a wide range of plant families. Deletions were found in the introns of both tRNA genes. Fourteen species had detectable deletions within the intron of trnI and four species within the intron of trnA. The occurrence of these deletions among the various plant families suggests that the events leading to the formation of these deletions occurred independently many times during the evolution of higher plants. Analysis of the amplified PCR products from the trnI intron suggests that these independent deletions may not be random but appear to fall into two size classes. Several members of each class were cloned and sequenced and the end points of the deletions were mapped. The 3' ends of all deletions studied terminate within the same short region. The 5' ends of the deletions map to two different regions, giving rise to the two size classes. These two 5' deletion endpoint regions show some sequence similarity. Only two of the identified deletions contain directly repeated sequences at the deletion endpoints, a feature associated with homologous recombination. Our results suggest that within the trnI intron, there are preferred sites or "hotspots" for deletion formation involving a novel imprecise recombination mechanism. The significance of these sequences and possible mechanisms for deletion formation are discussed.