The possibility that the stimulation of hexose transport in human fibroblasts by phorbol myristate acetate (PMA), insulin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) is associated with phosphorylation of the glucose transporter has been investigated. The time and concentration dependencies of the stimulation of transport by these agents under conditions identical to those used for phosphorylation were determined. Each agent, when used at the concentration that resulted in the maximal increase in transport rate, elicited this effect within 30 min of exposure. The extent of stimulation ranged from 15 to 70%. For determination of phosphorylation of the glucose transporter, fibroblasts were incubated for 16 h with [32P]Pi and exposed to the agonist for 30 min; the transporter was then isolated from a detergent lysate of the cells by immunoprecipitation with a monoclonal antibody. Under these conditions, there was no phosphorylation of transporter in basal cells and only PMA caused detectable incorporation of phosphate into the transporter. Thus, it is unlikely that the stimulation of glucose transport by insulin, PDGF and EGF involve transporter phosphorylation.