Dictyostelium discoideum cells synthesize and secrete the chemoattractant cAMP within minutes after chemotactic stimulation. During development, this signal-relay process is instrumental in cell aggregation, pattern formation, and differentiation. Cyclic AMP is known to accumulate inside the cell before secretion. In this study we investigated the subcellular localization of the nascent cAMP. After chemotactic stimulation at 0 degrees C and subsequent accumulation of intracellular cAMP, the newly synthesized chemoattractant could be released by gently opening cells in two different ways. Both methods make the cytosolic compartment accessible, whereas intracellular compartments surrounded by a membrane remain largely intact. The first method involved rapid lysis by forced passage through a 5-micron pore-size Nuclepore filter. The second technique was electropermeabilization under carefully controlled conditions that ensured the formation of small, stable pores in the plasma membrane. These pores allowed the passage of small molecules, such as cAMP, but not of macromolecules. To confirm the selectivity for the plasma membrane of both methods, we showed that a typical vesicular cell compartment, the lysosome, remained intact. Both procedures immediately released all intracellularly accumulated cAMP. We interpret our results as strong evidence for accumulation of nascent cAMP in the cytosolic compartment rather than in a vesicular compartment before it is secreted. This implies that cAMP secretion takes place via a trans-membrane transport mechanism, rather than by exocytosis.