Neurotransmitter L-norepinephrine increased up to 8-fold the activity of choline acetyltransferase (CAT), the enzyme responsible for the synthesis of acetylcholine, in mouse spinal cord cells in culture grown for several days. The increase of CAT activity by L-norepinephrine was mediated by a beta-adrenergic receptor in the same manner as the response of intracellular cyclic AMP. Derivatives of cyclic AMP caused an increase of CAT activity to the level similar to that of L-norepinephrine. A cyclic AMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine (IBMX), enhanced the elevation of CAT activity by L-norepinephrine. These results indicate that L-norepinephrine stimulated the synthesis of CAT molecules via the action of cyclic AMP. The pretreatment of cells with 5-fluoro-2'-deoxyuridine (FdU) markedly diminished the numbers of satellite cells and, in parallel, the responses of CAT activity to L-norepinephrine. The increase of cyclic AMP by L-norepinephrine was also reduced by pretreatment of the cells with FdU. In contrast, co-cultures of spinal cord with heart muscle markedly (30-fold) stimulated CAT activity both with and without pretreatment of FdU. The addition of L-norepinephrine and co-cultures with heart muscle showed an additive effect. These observations indicate that the stimulatory effect of L-norepinephrine on CAT activity is mostly, if not only, mediated via the interaction with satellite cells, and that the increase of CAT activity by L-norepinephrine is based on a mechanism different from that of co-cultures with heart muscle cells.