We have previously described an epidermal cell-derived thymocyte-activating factor (ETAF), which is produced by the murine PAM 212 keratinocyte cell line. ETAF appeared to be similar to macrophage-derived interleukin 1 (IL 1) in its biologic activities and biochemical characteristics. Both IL 1 and ETAF augment thymocyte proliferation, enhance lymphocyte production of interleukin 2 (IL 2), and are 15,000 m.w. polypeptides that are stable at pH 4 to 11 and from -70 degrees C to 60 degrees C. In this study we describe a quantitative microassay to obtain standardized assessment of ETAF activity, which enabled us to further define the characteristics of ETAF and its relationship to IL 1. Just as stimulated macrophages produce more IL 1 activity, Pam 212 keratinocyte production of ETAF activity was increased by stimulation with lipopolysaccharide (LPS) or silica. Increased levels were also obtained by mechanical disruption of confluent monolayers of keratinocytes and by blocking proliferation of the Pam 212 cells with hydroxyurea at the G1/S interphase. These observations in conjunction with a concomitant decrease in keratinocyte viability suggest that "injurious" stimuli that prolong the G1 phase of the cell cycle factor ETAF production. ETAF, like murine IL 1, has an isoelectric point of 5.2. The same subpopulations of PNA-thymocytes that respond to PHA and IL 1 are responsible for the enhanced proliferative response to ETAF. Furthermore, as in the case of IL 1, PNA- Lyt-2- thymocytes were most responsive to ETAF, but not PNA+ LYt-2+ thymocytes. Finally, ETAF activity, like IL 1, appears to be a mitogenic signal for fibroblasts. Although produced by different cell types, these observations continue to support the view that ETAF may be identical or closely related to IL 1.