The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Microfibril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags.