Ligand-induced oligomerization of receptors is a key step in initiating growth factor signaling. Nevertheless, complex biological responses often require additional trans-signaling mechanisms involving two or more signaling cascades. For cells of neuronal origin, it was shown that neurotrophic effects evoked by nerve growth factor or other neurotrophins depend highly on the cooperativity with cytokines that belong to the transforming growth factor beta (TGF-beta) superfamily. We found that rat pheochromocytoma cells, which represent a model system for neuronal differentiation, are unresponsive to TGF-beta1 due to limiting levels of its receptor, TbetaRII. However, stimulation with nerve growth factor leads to activation of the Smad pathway independent of TGF-beta. In contrast to TGF-beta signaling, activation of Smad3 by nerve growth factor does not occur via phosphorylation of the C-terminal SSXS-motif, but leads to heteromeric complex formation with Smad4, nuclear translocation of Smad3 and transcriptional activation of Smad-dependent reporter genes. This response is direct and does not require de novo protein synthesis, as shown by cycloheximide treatment. This initiation of transcription is dependent on functional tyrosine kinase receptors and can be blocked by Smad7. These data provide further evidence that the Smad proteins are not exclusively activated by the classical TGF-beta triggered mechanism. The potential of NGF to activate the Smad pathway independent of TGF-beta represents an important regulatory mechanism with special relevance for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those that are TGF-beta-resistant.