Abstract MicroRNAs (miRNA) are a group of short noncoding RNAs that regulate gene expression at the posttranscriptional level. They are involved in many biological processes, including development, differentiation, apoptosis, and carcinogenesis. Because miRNAs may play a role in the initiation and progression of cancer, they comprise a novel class of promising diagnostic and prognostic molecular markers and potential drug targets. By applying an LNA-enhanced microarray platform, we studied the expression profiles of 955 miRNAs in the NCI-60 cancer cell lines and identified tissue- and cell-type–specific miRNA patterns by unsupervised hierarchical clustering and statistical analysis. A comparison of our data to three previously published miRNA expression studies on the NCI-60 panel showed a remarkably high correlation between the different technical platforms. In addition, the current work contributes expression data for 369 miRNAs that have not previously been profiled. Finally, by matching drug sensitivity data for the NCI-60 cells to their miRNA expression profiles, we found numerous drug–miRNAs pairs, for which the miRNA expression and drug sensitivity profiles were highly correlated and thus represent potential candidates for further investigation of drug resistance and sensitivity mechanisms. Mol Cancer Ther; 10(3); 375–84. ©2011 AACR.