A biochemical procedure is described to purify the T-cell mitogen in the supernatant of cultured Mycoplasma arthritidis organisms. The mitogenic material was bound on an affigel blue column. The eluate of this column was then acylated at 0 degrees C for 1.5 h and subsequently chromatography on a Sepharose Cl 6B and a Superose 12HR column were performed. SDS-PAGE showed a major band at MW 26,000 and some minor bands at 50,000. With this material biological tests were performed, including induction of lymphoproliferation and interferon induction in murine spleen cell cultures. Purified Mycoplasma arthritidis supernatant (MAS) vigorously stimulated spleen cell cultures of A/J, CBA, C3H/He, and DBA/2 mice, whereas a low-grade but definitive response was observed in C57BL/6 spleen cells. Cultures of Balb/c nu/nu mice, in contrast to those of their euthymic littermates, were non-reactive. When induction of interferon was tested, a marked response to purified MAS was observed in CBA and C3H/HeJ spleen cell cultures, whereas C57BL/6 spleen cells were non-reactive.