We investigated the subcellular distribution of secretogranins I, II (Sg I, Sg II), and prolactin (PRL) by double immunogold electron microscopy in GH3B6 rat pituitary tumor cells grown in different culture conditions and in normal PRL cells in adult male rat anterior pituitary. Co-localization of Sg I or Sg II with PRL was observed in most secretory granules in GH3B6 cells and normal PRL cells, except for some secretory granules containing only Sgs in GH3B6 cells and containing only PRL in normal PRL cells. In GH3B6 cells treated with thyrotropin-releasing hormone (TRH) for 2 hr, the newly formed small secretory granules within the Golgi zone contained preferentially immunoreactive PRL. Interestingly, when co-localized with PRL, Sgs (particularly Sg I) were observed at the periphery of the matrix of secretory granules in GH3B6 cells as well as in normal PRL cells, suggesting their possible interaction with the secretory granule membrane. The present study indicates a heterogeneous subcellular distribution of PRL, Sg I, and Sg II in individual secretory granules of GH3B6 cells and of normal PRL cells, pointing out the formation of different types of aggregates during the condensation of secretory products in these two PRL cell models.