Glutamate is an attractant stimulus to Paramecium tetraurelia. It causes a hyperpolarization of the cell and smooth, relatively fast swimming that is characteristic of hyperpolarizing stimuli. We show here that by 1-30 seconds of stimulation, glutamate increases intracellular cAMP. Interestingly, other attractant stimuli, such as acetate and NH4Cl, that similarly hyperpolarize the cell do not induce an increase in cyclic AMP observable at 30 seconds. In order to determine whether the changes in cyclic AMP could be rapid enough to participate in stimulation as compared to slower processes such as adaptation, rapid kinetic measurements of cyclic AMP were made on whole cells by quenched-flow. We found that, in cells stimulated with glutamate, intracellular cyclic AMP increases by 30 mseconds and peaks at about sevenfold over basal levels by 200 mseconds. Cyclic GMP does not change relative to basal levels over rapid or slower time courses of glutamate stimulation. An antagonist of glutamate, IMP, depolarizes the cells and decreases intracellular cyclic AMP by approx. 50% and slightly increases cyclic GMP. Results of behavioral tests of cells treated with protein kinase inhibitors also suggest that cyclic AMP is part of the signal transduction pathway for glutamate, but not for other attractant stimuli. These studies are the first demonstration of a possible role for cyclic nucleotide second messengers in an attractant chemosensory transduction pathway in Paramecium.