The axonal guidance and outgrowth in retinal neurons were investigated in cultures of pure retinal neurons (control) or in cocultures with heterologous BC3H-1 cells. Under control conditions, only about 10% of retinal neurons developed axons; coculturing with BC3H-1 cells induced early axonal outgrowth and guidance to BC3H-1 cells in most amacrine neurons. Both mechanisms were dependent on laminin and neural cell-adhesion molecules (N-CAMs) released by BC3H-1 cells, because they were prevented by antibodies directed against these molecules. The protein kinase C (PKC) inhibitor, staurosporine, reduced the effect of laminin on amacrine axonal outgrowth, suggesting that this effect was mediated by PKC. The occurrence of structures resembling synaptic boutons and the expression of synaptophysin at the amacrine axon ends of heterologous connections suggested that amacrine axons establish true synaptic contacts rather than simply overlapping with the BC3H-1 cells. In contrast to the heterologous contacts with BC3H-1 cells, the amacrine-amacrine axonal contacts observed in the cocultures were independent of laminin and N-CAM. Axonal outgrowth occurred in about 10% of the photoreceptors and was not affected by BC3H-1 cells or by substratum pretreatment with laminin or N-CAM. These results show that different mechanisms affect axonal outgrowth and guidance in amacrine and photoreceptor neurons in vitro, and they suggest that similar mechanisms could contribute to the development of the scaffold of axon pathways in the retina in vivo.