AbstractCyclin dependent kinase (Cdk) 5, an atypical member of the Cdk family, plays a fundamental role in the development of the nervous system, and may also be involved in the pathogenesis of certain neurodegenerative diseases. Further, Cdk5 is activated by the specific regulatory proteins p39, p35, or p25 rather than cyclins, and in contrast to other members of the Cdk family is not involved in the progression of the cell cycle. A three‐dimensional computer model of Cdk5‐p25‐ATP has been generated previously [Chou et al., Biochem Biophys Res Commun 1999;259:420–428], providing a structural basis for the study of the mechanisms of Cdk5 activation. To assess the predicted ATP and p25 binding domains at the N‐terminal of Cdk5, two mutants of Cdk5 were prepared in which amino acids 9–15 (Δ9–15) or 9–47 (Δ9–47) were deleted. The results of these studies clearly demonstrate that an N‐terminal loop and the PSSALRE helix are indispensable for Cdk5‐p25 interactions, and amino acids 9–15 are necessary for ATP binding but are not involved in Cdk5‐p25 interactions. Predicted models of Δ9‐15 Cdk5 and Δ9‐47 Cdk5 were generated, and were used to interpret the experimental data. The experimental and molecular modeling results confirm and extend specific aspects of the original predicted computer model, and may provide useful information for the design of highly selective inhibitors of Cdk5, which could be used in the treatment of certain neurodegenerative conditions. Proteins 2002;48:447–453. © 2002 Wiley‐Liss, Inc.