Thirty healthy blood donors, 15 workers from horse-breeding farms, 69 human immunodeficiency virus (HIV)-negative persons at risk for HIV infection, 125 HIV-infected subjects without Rhodococcus equi infection, and nine HIV-infected patients with Rhodococcus equi pneumonia were evaluated in order to detect serum antibodies to Rhodococcus equi precipitate-soluble antigen by an enzyme immunoassay (EIA). Whereas EIA values for healthy donors, horse farm workers, individuals at risk for HIV infection, and HIV-positive subjects without Rhodococcus equi infection were comparable, HIV-infected patients with rhodococcal disease had significantly higher Rhodococcus equi antibody levels (p < 0.0001). The clinical outcome of Rhodococcus equi pneumonia was more severe in subjects who had low levels of specific antibodies, whereas patients who recovered had elevated Rhodococcus equi antibody levels over time. Immunoblot studies showed that both Rhodococcus equi-infected patients and foals recognized a protein band of approximately 60 kDa in the Rhodococcus equi precipitate-soluble antigen. On the other hand, the Rhodococcus equi-infected patients did not react with the diffuse 15 to 17 kDa virulence-associated proteins that represent important virulence factors both in mice and horses.