A secreted 450-kDa complex of proteins called counting factor (CF) is part of a negative feedback loop that regulates the size of the groups formed by developing Dictyostelium cells. Two components of CF are countin and CF50. Both recombinant countin and recombinant CF50 decrease group size in Dictyostelium. countin- cells have a decreased cAMP-stimulated cAMP pulse, whereas recombinant countin potentiates the cAMP pulse. We find that CF50 cells have an increased cAMP pulse, whereas recombinant CF50 decreases the cAMP pulse, suggesting that countin and CF50 have opposite effects on cAMP signal transduction. In addition, countin and CF50 have opposite effects on cAMP-stimulated Erk2 activation. However, like recombinant countin, recombinant CF50 increases cell motility. We previously found that cells bind recombinant countin with a Hill coefficient of approximately 2, a KH of 60 pm, and approximately 53 sites/cell. We find here that cells also bind 125I-recombinant CF50, with a Hill coefficient of approximately 2, a KH of approximately 15 ng/ml (490 pm), and approximately 56 sites/cell. Countin and CF50 require each other's presence to affect group size, but the presence of countin is not necessary for CF50 to bind to cells, and CF50 is not necessary for countin to bind to cells. Our working hypothesis is that a signal transduction pathway activated by countin binding to cells modulates a signal transduction pathway activated by CF50 binding to cells and vice versa and that these two pathways can be distinguished by their effects on cAMP signal transduction.