The pituitary hormone prolactin (PRL) plays an important role in mammary gland development. It was also suggested to contribute to breast cancer progression. In vivo data strongly supported a crucial role of PRL in promoting tumour growth; however, PRL demonstrated only a weak, if any, pro-proliferative effect on cancer cells in vitro. Several recent studies indicated that PRL action in vivo may be influenced by the hormonal milieu, e.g. other growth factors such as 17beta-oestradiol (E(2)). Here, we explored the potential interplay between PRL and E(2) in regulation of gene expression and cell growth. PRL alone induced either a weak or no proliferative response of T47D and BT-483 cells respectively, while it drastically enhanced cell proliferation in E(2)-stimulated cultures. Affymetrix microarray analysis revealed 12 genes to be regulated by E(2), while 57 genes were regulated by PRL in T47D cells. Most of the PRL-regulated genes (42/57) were not previously described as PRL target genes, e.g. WT1 and IER3. One hundred and five genes were found to be regulated upon PRL/E(2) co-treatment: highest up-regulation was found for EGR3, RUNX2, EGR1, MAFF, GLIPR1, IER3, SOCS3, WT1 and AREG. PRL and E(2) synergised to regulate EGR3, while multiple genes were regulated additively. These data show a novel interplay between PRL and E(2) to modulate gene regulation in breast cancer cells.