A 3.1-kb intergenic DNA fragment located between the psi beta-globin and delta-globin genes in the beta-globin gene cluster was cloned from gorilla, orangutan, rhesus monkey, and spider monkey, and the nucleotide sequence of each fragment was determined. The phylogeny of these four sequences, together with two previously published allelic sequences from humans and one from chimpanzee, was constructed, and the accumulation of mutations in the region was analyzed. The sites of base substitutions are not evenly distributed within the region: two Alu repeats have accumulated 0.21 + 0.02 substitutions/site with 0.15 + 0.008 substitutions/site in the remainder of the fragment. The occurrence of substitutions at neighboring sites is more frequent than would be expected if they were independent. The observed excesses disappear when ancestral -CG- dinucleotide sites are excluded. The phylogenetic relationships of the sequences indicate that the human sequence shares a most recent coancestor with the chimpanzee sequence. The data also show that great apes have accumulated fewer mutations in this part of the genome than has the rhesus monkey. The relative rates of accumulation of 12 kinds of nucleotide substitution in the region during primate evolution are asymmetric in the DNA strands. From these rates of accumulation, the origin of a simple stretch of sequence near the 3' end of the 3.1-kb fragment was deduced to be a sequence comprising 50% T and 50% C on one strand. The two oppositely oriented Alu sequences in the 3.1-kb region were inserted at their present positions before the divergence of the New-World monkeys from other lineages. Our analysis shows that the nucleotide sequences of the two Alu repeats in spider monkey are unexpectedly similar both to each other and to the deduced ancestral sequence of Alu repeats. The data suggest that there has been some type of recombinational event between the spider monkey Alu repeats but that it was not a simple gene conversion.