Positive transcription elongation factor b (P-TEFb) controls the fraction of initiated RNA polymerase II molecules that make full length transcripts. This important factor is a heterodimer of cyclin-dependent kinase 9 (Cdk9) and one of four cyclin partners, cyclin T1, T2a, T2b or K. There are two isoforms of Cdk9 in mammalian cells, Cdk9(42) and Cdk9(55). Cdk9(55) has a 117 residue amino terminal extension not present in Cdk9(42). An expression vector with a tetracycline-responsive promoter driving FLAG-tagged Cdk9(55) and a HeLa 37 Tet-Off cell line were constructed. FLAG-tagged Cdk9(55) was inducibly expressed and was found to be localized to the nucleus by immunofluorescence. Western analysis of murine tissues showed that the relative abundance of the two forms of Cdk9 varied across different tissues with liver having more Cdk9(55) than Cdk9(42). During adaptation of primary rat hepatocytes to culture the ratio of the two forms of Cdk9 changed. Initially, Cdk9(55) was the predominate form, but as the cells began to enter the cell cycle Cdk9(42) became the major form. During this change, expression of Cdk9(42) was induced, while Cdk9(55) remained relatively constant.