The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins-histone H2B-GFP and GFP-HP1alpha-before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.-Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins.