Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA (cpDNA) in order to study sequence-length variation in the Crassulaceae and in related species. Several length mutations were observed that are of diagnostic value for evolutionary relationships in the Crassulaceae and the Saxifragaceae. Length variation and sequence divergence in the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene among 15 species were studied in detail by nucleotide-sequence analysis. A total of 50 insertion/deletion mutations were observed, accounting for a spacer-length variation in the range of 228-360 bp. Eighteen short direct repeat motifs (4-11 bp) and two inverted repeat motifs (7-11 bp) were found to be associated with length variation. Phylogenetic analysis of the sequence data indicated a pattern of relationships that was largely consistent with a previous analysis of cpDNA restriction-site variation. Evaluation of the level of homoplasy in insertion/deletion mutations within a phylogenetic framework revealed that only 1 out of 34 length mutations longer than 2 bp must have had multiple origins. The feasibility of the noncoding chloroplast DNA regions for molecular evolutionary studies is discussed.