Coupling of the three known alpha1-adrenergic receptor (alpha1-AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing alpha1A-, alpha1B-, and alpha1D-ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2+ level in these cells in a manner dependent on receptor density. However, alpha1A-ARs activated these second messenger responses more effectively than alpha1B-ARs, whereas alpha1D-ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three alpha1-AR subtypes, although alpha1A- and alpha1B-ARs caused larger ERK activation than did alpha1D-ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in alpha1A- and alpha1B- but not alpha1D-transfected cells and activated c-Jun NH2-terminal kinase (JNK) only in alpha1A-transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in alpha1A-transfected PC12 cells. NE caused neurite outgrowth only in alpha1A-expressing PC12 cells, but not in alpha1B- or alpha1D-transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that alpha1A-ARs activate all three MAPK pathways, alpha1B-ARs activate ERKs and p38 but not JNKs, and alpha1D-ARs only activate ERKs. Only the alpha1A-AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.