Thapsigargin, a protein kinase C-independent tumor promoter, can negatively regulate the epidermal growth factor (EGF) receptor through inhibition of high affinity EGF binding and EGF-stimulated tyrosine kinase activity. In contrast to activators of protein kinase C, thapsigargin does not induce significant phosphorylation of threonine 654. However, thapsigargin does stimulate phosphorylation of the EGF receptor at other serine and threonine residues. We now identify threonine 669 as the major site of phosphorylation on the EGF receptor resulting from thapsigargin treatment. These results raise the possibility that phosphorylation of threonine 669 may mediate changes in the binding and kinase state of the EGF receptor.