The sphingomyelin (SM) cycle is an emerging pathway of signal transduction that plays a role in the control of cell growth, cell differentiation, and apoptosis. During earlier investigation of SM pools hydrolyzed in the SM cycle, we examined the effects of the fungal macrolide brefeldin A (BFA) on cellular levels of SM in HL-60 leukemia cells. We found that BFA induced up to 20-25% hydrolysis of SM. Here we show that this BFA-sensitive SM pool corresponds to the pool of SM hydrolyzed by a previously discovered activator of the SM cycle, 1,25-dihydroxyvitamin D3. BFA was also able to induce the biological end points of SM cycle activation: growth inhibition and differentiation. Reciprocally, ceramide inhibited the secretion of 35S-labeled proteins from HL-60 cells and induced a subset of effects of BFA on organelle morphology. Since a ceramide-activated protein phosphatase has been previously suggested as a direct in vitro target of ceramide action, the effects of modulators of protein kinases and phosphatases were examined. Okadaic acid enhanced protein secretion and was able to oppose the effects of both ceramide and BFA on organelle morphology. Dioctanoylglycerol and phorbol myristate acetate, known activators of protein kinase C, were also found to oppose the inhibitory actions of ceramide on secretion. These studies identify BFA as an activator of the SM cycle, with ceramide as a potential mediator of some of the effects of BFA. Additionally, taken with the effects of the PKC activators, these studies suggest that constitutive protein secretion is not a default pathway but is subject to regulation by processes of signal transduction.