The response of the Na efflux in unpoisoned barnacle fibers to 10 mM theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10 mM theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500 mM EGTA completely abolishes the biphasic action of 10 mM theophylline. External application of 10 mM theophylline following removal of external Ca2+ fails to bring about a biphasic effect. Ca2+ restoration, however, results in a moderate rise in the Na efflux. External application of 10 mM theophylline stimulates the Na efflux into Ca2+-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10 mM theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10 mM theophylline. Injection of cAMP into ouabain-poisoned fibers, following internal application of Corbin's inhibitor and external application of 10 mM theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10 mM theophylline, fails to reduce the Na efflux. Fibers injected with 1 mM and 100 mM EGTA and exposed to 10 mM theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10 mM. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10 mM theophylline. Theophylline (10 mM) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3 mM-HEPES ASW or 10 mM-Ca2+ -3mM-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa(pCa=-log10[Ca2+]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.