Recently, a technique was described for amplification of Rhodococcus equi-specific chromosomal and vapA DNA from blood and tracheal wash fluids. It was hypothesized that this technique would be more sensitive than standard culture techniques or serology for diagnosis of R. equi pneumonia in foals. Tracheal wash fluid, nasal swabs, whole blood samples, and serum samples from 56 foals with pneumonia were analyzed. Final clinical diagnosis was determined by the attending clinician on the basis of final interpretation of all available information about each foal, including clinical presentation, diagnostic test results, response to therapy, and outcome. Clinical diagnosis was used as a final reference standard for calculation of sensitivity, specificity, and predictive values for PCR, serology using an agar gel immunodiffusion test, and tracheal wash fluid culture. PCR of tracheal wash fluid using primers that recognized the vapA virulence plasmid of R. equi had a diagnostic sensitivity of 100% and specificity of 90.6%. Sensitivity and specificity were 57.1 and 93.8%, respectively, for standard microbiologic culture of tracheal wash fluid and 62.5 and 75.9%, respectively, for serology. PCR of tracheal wash fluid is more sensitive and specific for diagnosis of R. equi pneumonia than are other available diagnostic tests.