We found novel development rescuing factors (DRFs) secreted from developing Dictyostelium cells, by using a mutant (erkB-) which is missing MAP-kinase ERK2 as a test strain for bioassay. The mutant erkB- fails to undergo multicellular morphogenesis due to impaired cAMP signaling. However, such developmental defect can be restored by the presence of low-molecular weight DRFs that are secreted from developing wild-type cells. We previously showed that DIF-1 (Differentiation-Inducing Factor 1 for stalk cells) possesses this activity, indicating a newly discovered role of DIF-1. Surprisingly, however, the mutant dmtA-, which is incapable of DIF-1 synthesis still exerts a strong inducing activity of the multicellular morphogenesis of erkB-. After analysis of HPLC fractions of conditioned media prepared from both wild type Ax2 and dmtA- strains revealed that both strains secrete at least two novel DRF activities with DIF-like mobility. However, these activities were not derived from other DIFs such as DIF-2 and DIF-3. Identification of these DRFs found in this study would provide insight into the mechanism by which the development of the erkB- mutant is restored and how these factors act in the normal development of Dictyostelium.