Mammalian heterochromatin protein 1 (HP1α, HP1β, HP1γ subtypes) and transcriptional intermediary factor TIF1β play an important role in the regulation of chromatin structure and function. Here, we investigated the nuclear arrangement of these proteins during differentiation of embryonal carcinoma P19 cells into primitive endoderm and into the neural pathway. Additionally, the differentiation potential of trichostatin A (TSA) and 5-deoxyazacytidine (5-dAzaC) was studied. In 70% of the cells from the neural pathway and in 20% of TSA-stimulated cells, HP1α and HP1β co-localized and associated with chromocentres (clusters of centromeres), which correlated with clustering of TIF1β at these heterochromatic regions. The cell types that we studied were also characterized by a pronounced focal distribution of HP1γ. The above-mentioned nuclear patterns of HP1 and TIF1β proteins were completely different from the nuclear patterns observed in the remaining cell types investigated, in which HP1α was associated with chromocentres while HP1β and HP1γ were largely localized in distinct nuclear regions. Moreover, a dispersed nuclear distribution of TIF1β was observed. Our findings showed that the nuclear arrangement of HP1 subtypes and TIF1β is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes.