Purified endogenous pyrogen (EP), isolated in one laboratory from stimulated rabbit peritoneal exudate cells (PEC), was compared with purified lymphocyte activating factor (LAF), isolated in another laboratory from stimulated rabbit alveolar macrophages (AM). Both EP and LAF occurred in two forms, one with an isoelectric point (pI) of 7.3 to 7.4, the other with a pI of 4.6 to 4.7. Both forms of EP and LAF had m.w. of 13,000 to 16,000 daltons as judged by gel filtration. Both forms of EP had LAF activity, and both forms of LAF were pyrogenic. The pI 7.3 EP, which is known to have an -SH group essential for its biologic activity, bound to Thiol Sepharose columns and could be eluted with mercaptoethanol. The pI 7.3 LAF behaved in exactly the same way. Furthermore, antisera from three different goats were available that completely blocked the pyrogenicity of pI 7.3 EP in vivo; these sera also blocked the pI 7.3 LAF activity in vitro. The pI 4.6 EP and LAF did not bind to Thiol Sepharose columns, nor were they inhibited by any of the antisera that blocked the pI 7.3 EP and LAF. Moreover, isoelectric focusing of pI 4.6 EP in very shallow gradients revealed microheterogeneity with sharp peaks of EP activity observed at pH 4.6 and 4.7. Analysis for LAF activity showed an identical microheterogeneity. These results are consistent with the idea that EP and LAF are identical.