ABSTRACT Cyclic AMP concentrations have been measured in a number of different cell types under a variety of culture conditions in an attempt to define the relationship between the endogenous concentration of cyclic AMP and cell mobility. In previous work it was shown that agents that increase the intracellular concentration of cyclic AMP can effectively suppress cell movement. In Balb/c 3T3 cells, which have a very low mobility in cellular aggregates, the intracellular concentration of cyclic AMP was elevated only transiently soon after the formation of the three-dimensional cell masses. In contrast, in the highly mobile virally transformed counterpart of Balb/c 3T3 cells, called SVT- 2, the concentration of cyclic AMP was relatively low soon after the cell masses were formed, but later rose to a level that was higher than that in Balb/c 3T3 cells. Using NIL B cells, SV40- transformed NIL B cells, and several lines of tumour cells derived from NIL B cells, it was found that the average intracellular concentration of cyclic AMP did not vary significantly from one population of cells to another. Finally, the intracellular concentration of cyclic AMP was measured in chick embryo ventricle cells. The mobility of these cells had previously been found to decrease as embryonic development progressed; furthermore, it had been shown that dibutyryl cyclic AMP plus theophylline produced nearly complete inhibition of their movement in cell masses. In the series of experiments reported here we found that the endogenous concentration of cyclic AMP in aggregates and fragments of chick embryo ventricle cells decreases as development proceeds; these data are consistent with preliminary experiments reported by other investigators. In a separate set of experiments, the intracellular concentration of cyclic AMP was measured in cells that had been cultured in a medium containing 1 · 2 mM-dibutyryl cyclic AMP plus 1 · 0 mM-theophylline. This drug treatment has previously been shown to inhibit the movement of cells both in aggregates and in monolayers; it also produces striking effects on cell shape and ultrastructure. In aggregates of chick embryo ventricle cells, treatment with these drugs resulted in increases in the intracellular concentrations of cyclic AMP from approximately 10 picomol/mg protein to approximately 500 picomol/mg protein. In Balb/c 3T3 and SVT-2 cells this treatment increased cyclic AMP con centrations from 3 · 7 to 160 and from 6 · 4 to 470 picomol/mg protein, respectively.