This study explored cooperation between the retinoic acid (RA) and protein kinase C (PKC) pathways during differentiation of the multipotential human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1). We report here that, compared to RA treatment alone, RA combined with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the regulated expression of the immunophenotypic differentiation markers SSEA-3, a globo-series carbohydrate, and the ganglio-series carbohydrate antigens GD2 and GD3. Northern analysis and transient transfection assays revealed that TPA co-treatment augmented the RA-induced expression and activation of the RA nuclear receptor-β (RAR-β), one early marker of RA response in NT2/D1 cells. This finding was extended with transient co-transfection experiments using a PKC-α expression vector which revealed that the PKC pathway can augment the activation of RAR-β by RA. These experiments establish PKC as a modulator of RAR-β expression in NT2/D1 cells. Similarly, experiments showed that RA can modulate activation of the PKC-responsive AP- 1 complex, a transcription factor rapidly activated by TPA. Northern analysis and transient transfection assays revealed that, compared to TPA treatment alone, RA and TPA augmented the expression and transcriptional activity of AP-1 in NT2/D1 cells. In contrast, transient transfection assays revealed no cooperative effect between RA and TPA in HeLa cells, indicating that this effect in NT2/D1 cells is cell type-specific. In summary, these studies show that stimulation of the PKC second messenger pathway can modulate tumor differentiation and transcriptional activation of a retinoid receptor associated with RA response. © 1993, International Society of Differentiation. All rights reserved.