A mitogenic anti-CD3 ("T3") monoclonal antibody (64.1), that stimulates polyclonal T cell activation by a mechanism believed to be similar to antigen via binding to the T cell receptor complex, was utilized in soluble (SOL) and Sepharose-bound (SEPH) forms to dissect the role of accessory cells (AC) and interleukin 1 (IL 1) in supporting T cell activation. The T cell activation pathway was dissected into "early" events including expression of interleukin 2 receptors (IL 2R), increased RNA content, IL 2 release, and "late" (DNA synthesis) events. Unseparated peripheral blood mononuclear cells progressed through all stages of activation when stimulated by either form of 64.1. Stringent AC depletion by plastic adherence, nylon wool adherence, and L-leucine methyl ester (selectively lyses AC) prevented early and late T cell responses to either form of 64.1. The addition of highly purified IL 1 replenished both early and late T cell responses to SEPH-64.1 but not to SOL-64.1. Although SOL-64.1 stimulation of purified T cells induced modulation of the CD3 complex, only SEPH-64.1 induced IL 1 responsiveness, and exogenous IL 1 was then able to support synthesis of RNA, secretion of IL 2, expression of IL 2R, and ultimately, DNA synthesis. Therefore, the stages of early T cell activation owing to stimulation of the CD3-T cell receptor complex and IL 1 responsiveness have been dissected.