Thirty formaldehyde-fixed, paraffin-embedded endoscopic biopsy specimens from the colon and rectum of 25 patients with acquired immunodeficiency syndrome were examined using a [35S]HIV-RNA in situ hybridization procedure. Nine of the specimens contained cells that bound significant amounts of probe. Cells were considered positive if more than 50 grains of silver (over background) per 200 micron 2 were seen over cells that did not stain with eosin. Most of the positive cells resembled macrophages, although cells with condensed nuclei resembling lymphocytes were found. No epithelial cells expressing viral RNA were detected. Formaldehyde-fixed eosinophils gave spurious signals that could be reduced with sulfhydryl modifying agents. HIV-1 may be disseminated in the lamina propria of the gut at low concentrations in some patients but may not be detectable in others. The lower gut lining may be both a portal of initial infection with HIV and a target of disseminated HIV infection.