The ability to productively infect fresh normal human peripheral blood mononuclear leukocytes with HTLV-III was improved by supplementing cell culture medium with either the gonadal steroid, chorionic gonadotropin, or insulin, and more substantially with the adrenocortical steroid, hydrocortisone. Several other sex hormones and another corticosteroid, dexamethasone, had no significant effect. In addition, the isolation of HTLV-III from lymphocytes established in culture from patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), and healthy, at-risk donors was greatly facilitated by the inclusion of hydrocortisone in cell culture media. In 13/20 primary cell cultures tested from AIDS and ARC patients from whom virus was isolated, the amount of virus produced was elevated from low to easily detectable levels in those containing hydrocortisone. In 3/20 specimens tested, virus was detected and isolated from cell cultures supplemented with hydrocortisone but was undetectable in those lacking the hormone. These experiments demonstrate that hydrocortisone, a readily available, inexpensive supplement to cell culture media, can facilitate the detection and isolation of HTLV-III. These studies, furthermore, suggest a role for corticosteroids and possibly gonadal steroids in the modulation of virus expression and/or release and suggest that the viral inductive capacity of these and other compounds should be considered as they are evaluated for clinical use.