Our previous studies showed that relaxin promotes differentiation of MCF-7 breast adenocarcinoma cells. In the current investigation, we aimed to elucidate whether the effect of the hormone is potentiated when MCF-7 cells are grown together with myoepithelial cells, thus creating a microenvironment reminiscent of the organised tissue architecture of the mammary parenchyma in vivo. The findings obtained reveal that most MCF-7 cells cultured alone have an undifferentiated, blast-like phenotype, only a minority showing a more differentiated phenotype with more organelles and rudimentary intercellular junctions. When co-cultured with myoepithelial cells more MCF-7 cells acquire ultrastructural features consistent with a more differentiated phenotype, such as a rich organellular complement, apical microvilli and intercellular junctions. When relaxin was added to the co-cultures, the ultrastructural signs of differentiation could be observed in even more MCF-7 cells and became more pronounced than in the absence of the hormone, judged by the appearance of a clear-cut polarisation of cytoplasmic organelles, an almost continuous coat of apical microvilli and numerous intracellular pseudolumina.