1. The effects of gamma-interferon (gamma-IFN), retinoic acid (RA), and cytosine arabinoside (ARA-C) on the growth, morphology, and phenotype of the human neuroblastoma (NB) cell lines, LAN-1 and GI-ME-N, have been extensively tested. 2. RA, gamma-IFN, and ARA-C induced a dose-dependent morphological differentiation and growth inhibition, without affecting cell viability. Cells exposed to 10(-6) M RA or 1000 U/ml gamma-IFN significantly decreased their growth rate within the first 24 and 48 hr of culture, respectively. Cells became smaller and polygonal and sprouted long cellular processes with varicosities along their courses. In contrast, ARA-C-differentiated cells were larger and flattened, with few elongated dendritic processes. 3. Analysis of membrane and cytoskeletal markers by immunofluorescence and Western blot showed several changes in NB-specific antigen expression after 5 days of treatment with all inducing agents. Analysis of labeled phosphatidylinositol metabolites from prelabeled cells showed, within 1 min of treatment with RA, a rapid decrease in inositol 1,4,5-trisphosphate and of 1,2-diacylglycerol levels. No changes in inositol phospholipid metabolism were observed in gamma-IFN- or ARA-C-treated cells. 4. We conclude that RA-induced decrease in phosphatidylinositol (PI) hydrolysis is not likely to be a consequence of the acquisition of a different phenotype, as its changes precede the acquisition of neuronal markers. In addition, gamma-IFN and ARA-C, both inducing a mature phenotype, did not affect PI hydrolysis. 5. Decreased PI hydrolysis seems to be sufficient, although not necessary, to commit NB cells to neuronal differentiation. Analysis of molecular mechanisms associated with NB cell differentiation may be helpful to clarify the potential of various biological agents in affecting the development of the neural cell.