The HER2 protooncogene encodes a growth factor receptor-like transmembrane protein tyrosine kinase (p185HER2) whose ligand remains to be fully characterized. The overexpression of p185HER2 is implicated in aggressive forms of breast and ovarian cancers. The role of p185HER2 in aggressive malignancy, as well as its cell surface localization, makes it an attractive target for therapeutic monoclonal antibodies. In this report we have studied the modulation of p185HER2 function with 2 monoclonal antibodies, termed 4D5 and 6E9, which bind the extracellular domain of p185HER2. 4D5 inhibited proliferation of p185HER2 overexpressing SK-BR-3 human breast carcinoma cells (ED50 of approximately 1 nM) but did not inhibit proliferation of cultured human breast carcinoma MCF7 cells, low expressors of p185HER2. Monoclonal antibody 6E9 does not inhibit the growth of either cell line. Antibody binding studies revealed 2 populations of p185HER2 molecules on SK-BR-3 cells: one of high abundance (approximately 2 x 10 (6) sites/cell) recognized by 4D5 (Kd approximately 6 nM) and the other of low abundance (2 x 10 (4) sites/cell) recognized by 6E9 (Kd approximately 0.1 nM). 4D5, in an agonistic manner, downregulated SK-BR-3 cell surface p185HER2, was internalized, and stimulated p185HER2 phosphorylation in intact cells. Phosphoamino acid analysis of p185HER2 derived from SK-BR-3 cells incubated with the 4D5 monoclonal antibody demonstrated increased tyrosine, serine and threonine phosphorylation. 4D5, on short term (5 min) exposure to SK-BR-3 cells, stimulated inositol lipid hydrolysis as evidenced by increased intracellular levels of inositol polyphosphates (InsP) and sn-1, 2-diacylglycerol (sn-1, 2-DAG). On longer (24 h) exposure to the cells, the antibody appeared to downregulate this signalling pathway since the intracellular levels of InsP and sn-1, 2-DAG decreased by 30 to 40%. 6E9 did not inhibit SK-BR-3 cell proliferation, downregulate surface p185HER2, stimulate receptor phosphorylation, or stimulate the second messenger pathway. Despite these agonistic properties, 4D5 was an inhibitor of SK-BR-3 cell proliferation at all concentrations tested (0.7 to 70 pM). The data suggest that 4D5 is a partial or weak agonist and thus may inhibit cell proliferation by mimicking ligand-like receptor downregulation.