Proteins that bind and stabilize single-stranded DNA are critical for proper DNA metabolism. In eukaryotes, the major single-stranded DNA-binding protein is replication protein A (RPA), an evolutionarily conserved heterotrimeric complex required for DNA replication, repair, and recombination. While much of the early work on RPA established its role in DNA replication, a great deal of attention is now being paid to the specific mechanisms by which RPA operates in recombination. As described in this review, significant insight has been gained from studies employing proteins purified from both yeast and human cells. Of particular importance, these analyses have revealed that RPA is centrally involved in the initiation of homologous recombination. Research into recombination and its influence by RPA is especially relevant to our understanding of disease development, as inappropriate chromosomal rearrangement is known to be associated with a number of human disorders.