Mammalian cell lines are currently employed as one of the main cellular factories for the expression of recombinant protein-based drugs. The establishment of high-producing cell lines typically begins with a heterogeneous starter population of cells, from which the highest producing cells are selected via empirical approaches. This approach is time consuming, and is likely to encounter natural upper limits imposed by the inherent biology of the cell lines in question. In an attempt to understand both the nature of the variability in populations of cells transfected with recombinant protein encoding DNA and the natural mechanisms of productivity limitation, we developed protocols for the detailed investigation of gene expression pathways in such cell lines. This novel approach was then applied to a set of clonal CHOK1 cell lines producing recombinant luciferase with varying productivities. Our results show that the initial limitation in these cell lines is at the transcriptional level, however in the highest producing cell line post-translational mechanisms affecting both protein turnover and protein folding become severely limiting. The implications for the development of strategies to engineer cells for enhanced recombinant protein production levels are discussed. Biotechnol. Bioeng. 2009;102: 1593–1602. © 2008 Wiley Periodicals, Inc.