Pyomelanin overproduction is a common phenotype among Pseudomonas aeruginosa isolates recovered from cystic fibrosis and urinary tract infections. Its prevalence suggests that it contributes to the persistence of the producing microbial community, yet little is known about the mechanisms of its production. Using transposon mutagenesis, we identified factors that contribute to melanogenesis in a clinical isolate of P. aeruginosa. In addition to two enzymes already known to be involved in its biosynthesis (homogentisate dioxygenase and hydroxyphenylpyruvate dioxygenase), we identified 26 genes that encode regulatory, metabolic, transport, and hypothetical proteins that contribute to the production of homogentisic acid (HGA), the monomeric precursor of pyomelanin. One of these, PA14-57880, was independently identified four times and is predicted to encode the ATP-binding cassette of an ABC transporter homologous to proteins in Pseudomonas putida responsible for the extrusion of organic solvents from the cytosol. Quantification of HGA production by P. aeruginosa PA14 strains missing the predicted subcomponents of this transporter confirmed its role in HGA production: mutants unable to produce the ATP-binding cassette (PA14-57880) or the permease (PA14-57870) produced substantially less extracellular HGA after growth for 20 h than the parental strain. In these mutants, concurrent accumulation of intracellular HGA was observed. In addition, quantitative real-time PCR revealed that intracellular accumulation of HGA elicits upregulation of these transport genes. Based on their involvement in homogentisic acid transport, we rename the genes of this operon hatABCDE.