The p100 coactivator, first identified as a coactivator of the Epstein-Barr virus-encoded transcription factor, EBNA-2, in cultured cells, interacts with a number of transcription factors. However, the role of p100 in animals is unclear. We found that the abundance of p100 is closely associated with the lactating state in mammary tissue of mice and cows. Using two antibodies against independent parts of the protein, p100 immunoreactivity was localised to mammary epithelial cells, and was enriched in both nuclei and endoplasmic reticulum/organelle fractions. Stimulation of beta-casein expression in cultured mammary epithelial cells was associated with an increase in abundance of the p100 protein. The relative abundance of p100 mRNA was not altered in mammary tissue throughout the gestation-lactation cycle, indicating that the abundance of p100 is altered by a post-transcriptional mechanism. Further work is required to clarify the function of p100 in mammary epithelial cells.