One emphasis of the Gibbs Conference on Biothermodynamics is the value of thermodynamic measurements for understanding behaviors of biological systems. In this study, the correlation between thermodynamic measurements of in vitro DNA binding affinity with in vivo transcription repression was investigated for two transcription repressors. In the first system, which comprised an engineered LacI/GalR homolog, mutational changes altered the equilibrium constant for binding DNA. Changes correlated with altered repression, but estimates of in vivo repressor concentration suggest a ≥25-fold discrepancy with in vitro conditions. In the second system, changes in ligand binding to BirA altered dimerization and subsequent DNA occupancy. Again, these changes correlate with altered in vivo repression, but comparison with in vitro measurements reveals a ~10-fold discrepancy. Further analysis of each system suggests that the observed discrepancies between in vitro and in vivo results reflect the contributions of additional equilibria to the transcription repression process.