Platelet activating factor (PAF)-stimulation of human B-lymphoblastoid cells results in the activation of microtubule associated protein 2-kinase (MAPK) and increases in intracellular calcium. Although increases in intracellular calcium induce MAPK activation in these cells, PAF can stimulate MAPK activation in the absence of detectable changes in intracellular calcium concentrations ([Ca2+]i). Treatment of the LA350 B-lymphoblastoid cell line with either pertussis toxin (PT) or cholera toxin (CT) blocked PAF-induced changes in [Ca2+]i. However, only PT blocked PAF-induced activation of MAPK as determined by shifts in the mobility of MAPK on immunoblots. In support of this finding, only PT but not CT blocked PAF-induced phosphorylation and activation of p90rsk, an event thought to be distal to MAPK activation. These results suggest that the PAF receptor is mediating MAPK activation through pathways separate from those mediating increases in intracellular calcium.