CDK9 associates with T-type cyclins and positively regulates transcriptional elongation by phosphorylating RNA polymerase II (RNAPII) and negative elongation factors. However, it is unclear whether CDK9 is required for transcription of most genes by RNAPII or alternatively plays a role regulating the expression of restricted subsets of genes. We have investigated the direct effects of inhibiting cellular CDK9 activity in global gene expression in human cells by using a dominant-negative form of CDK9 (dnCDK9). We have also compared direct inhibition of cellular CDK9 activity to pharmacological inhibition with flavopiridol (FVP), a CDK inhibitor that potently inhibits CDK9 and cellular transcription. Because of its presumed selectivity for CDK9, FVP has been previously used as a tool to infer the role of CDK9 on global gene expression. DNA microarray analyses described here show that inhibition of gene expression by FVP is consistent with global inhibition of transcription. However, specific inhibition of CDK9 activity with dnCDK9 leads to a distinctive pattern of changes in gene expression, with more genes being specifically upregulated (122) than downregulated (84). Indeed, the expression of many short-lived transcripts downregulated by FVP is not modulated by dnCDK9. Nevertheless, consistently with FVP inhibiting CDK9 activity, a significant number of the genes downregulated/upregulated by dnCDK9 are modulated with a similar trend by FVP. Our data suggests that the potent effects of FVP on transcription are likely to involve inhibition of CTD kinases in addition to CDK9. Our data also suggest complex and gene-specific modulation of gene expression by CDK9. J. Cell. Physiol. 222:200–208, 2010. © 2009 Wiley-Liss, Inc.