The Drosophila melanogaster fourth chromosome, believed to be nonrecombining and invariable, is a classic example of the effect of natural selection in eliminating genetic variation in linked loci. However, in a chromosome-wide assay of nucleotide variation in natural populations, we have observed a high level of polymorphism in a approximately 200-kilobase region and marked levels of polymorphism in several other fragments interspersed with regions of little variation, suggesting different evolutionary histories in different chromosomal domains. Statistical tests of neutral evolution showed that a few haplotypes predominate in the 200-kilobase polymorphic region. Finally, contrary to the expectation of no recombination, we identified six recombination events within the chromosome. Thus, positive Darwinian selection and recombination have affected the evolution of this chromosome.