beta 1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is localized both in the Golgi complex and on the cell surface. Most studies of surface GalTase function have focused on its role in cellular interactions; however, surface GalTase has also been suggested to function during cellular proliferation. Consistent with this hypothesis, a variety of GalTase-specific perturbants inhibit cell growth in vitro and in vivo. However, all of these studies have been limited to the use of exogenous reagents to perturb GalTase function. Furthermore, all of these perturbants inhibit cell growth, irrespective of whether they stimulate or inhibit GalTase enzyme activity. Therefore, it remains unclear whether surface GalTase delivers a growth inhibitory or growth stimulatory signal. In this study, we took a more direct approach to defining surface GalTase function during growth by examining its expression during the cell cycle and by molecularly altering its expression in stably transfected cell lines. The expression of GalTase was shown to be cell cycle specific, with the cell surface and intracellular GalTase pools displaying independent expression patterns. Furthermore, multiple, independent, stably transfected cell lines with reduced levels of cytoskeletally associated surface GalTase grew faster than control cells, whereas cell lines that over-expressed surface GalTase grew slower than controls. These observations directly support the concept that surface GalTase delivers a growth inhibitory signal. Evidence is presented suggesting that surface GalTase interacts with the epidermal growth factor (EGF) receptor, as suggested by others. The activity of the EGF receptor was shown to be directly proportional to the growth rate of the various GalTase-transfected cell lines. Thus, the expression of surface GalTase directly affects cell proliferation rate and may do so by modulating the ability of the EGF receptor to transduce EGF-dependent signals.