The effect of estrogen on prolactin (PRL) synthesis at a single-cell level was studied by in situ hybridization and immunohistochemistry. Long-term estrogen treatment increased PRL-containing cells from 10–20% of total cell population to 80–90%, as revealed by immunohistochemistry. PRL mRNA containing cells also increased in a similar fashion. Moreover, cytoplasmic PRL mRNA expressed as the number of silver grains per cell increased 4- to 5-fold by estrogen. These results suggest that long-term estrogen treatment causes not only PRL cell proliferation but also an increase in PRL mRNA in a single c