The preferred DNase I cleavage sites within the 160 bp tyrT DNA fragment were identified by studying the initial rate of cleavage of individual bonds. The results show that there is no correlation between the rate of cleavage and the identity of the dinucleotide sequence that is cleaved. Examination of the sequences surrounding the seven most rapidly cleaved bonds suggests that an A-T base-pair is preferred at the position three bases to the 5' side of the cleavage site. Preferential cleavage at such sites is consistent with predictions based on the recently obtained high resolution structure of a DNase I-octanucleotide complex. A statistical analysis of 54 additional preferred DNase I cleavage sites, using sequence data taken from published literature, confirms that DNase I exhibits a local sequence preference in addition to its relatively well characterized global structural specificity. Our analysis indicates preferential cleavage at the sequences 5'ATYAT--ATVN, where -- indicates the cleavage site, the notation AT indicates a preference for an A-T base-pair, and V indicates not-T. Comparative kinetic studies of the digestion of three deoxyoctanucleotides by DNase I quantitatively support the sequence preference inferred from the sequence analysis. Poor DNase I cleavage sites were also examined, and found to be characterized by the sequence motif 5'GCRR--TTY. Notably, poor cleavage sites characteristically contain G or C at position -3. While DNase I certainly does not cleave with an absolute sequence specificity, our studies reveal a distinct sequence preference in DNase I cleavage that has heretofore been unappreciated and uncharacterized.