OBJECTIVE: To investigate perturbations in downstream signaling pathway activation and potential resistance mechanisms to epidermal growth factor receptor (EGFR) or human epidermal growth factor receptor 2 (HER2) inhibition in cell line models of bladder cancer. METHODS: We undertook a structured screening approach by phosphokinase array, followed by validation steps, to detect activated downstream signaling pathway nodes after therapeutic inhibition of EGFR or HER2 in bladder cancer cell lines. RESULTS: Erlotinib treatment of RT112 cells induced phosphorylation of 9 activated phosphoprotein targets (p38 mitogen-activated protein kinase [MAPK] [Thr180/Tyr182], GSK-3alpha/beta [Ser21/9], MEK1/2 [Ser218/222, Ser222/226], Akt (protein kinase B) [Ser473], TOR [target of rapamycin] [Ser2448], Src [Tyr419], p27 [Thr198], p27 [Thr157], and PLCgamma-1 [Tyr783]), whereas STAT4 (signal transducer and activator of transcription 4) (Tyr693) phosphorylation was reduced. Of these, p38 MAPK phosphorylation was confirmed to occur in response to inhibition of either EGFR or HER2 signaling through multiple validation steps, including differing bladder cancer cell lines (RT112, UM-UC-3, and T24) and methods of receptor pathway inhibition (erlotinib, lapatinib, and siRNA depletion of EGFR or HER2). Chemical inhibition of p38 MAPK with SB203580 led to inhibition of proliferation in RT112, UM-UC-3, and T24 cell lines (IC50 20.85, 76.78, and 79.12 microM, respectively). Fractional effect analyses indicated a synergistic interaction for inhibition of cell proliferation when combining SB203580 with lapatinib. CONCLUSION: p38 MAPK is a potential therapeutic target in bladder cancer and this strategy warrants further development in this disease. It may also allow combination therapy strategies to be developed in conjunction with EGFR or HER2 inhibition.