The existence of bihormonal anterior pituitary (AP) cells co-storing growth hormone and either prolactin (mammosomatotrope) or gonadotropins (somatogonadotrope) has been described. These cells have been proposed to be involved in "paradoxical" secretion [secretion of an AP hormone induced by a non-related hypothalamic releasing factor (HRH) and transdifferentiation (a phenotypic switch between different cell types without cell division]. Here we combine calcium imaging (to assess HRH responsiveness) and multiple sequential immunoassay of the six AP hormones to perform a single-cell phenotypic study of multifunctional somatotropes, mammotropes and gonadotropes in the normal male and female mouse pituitaries. AP cell phenotypes differed from the classic view, showing multiple HRH-receptor expression and/or hormone storage. Mammosomatotropes represented only 5-6% of somatotropes and were poorly responsive to HRHs, suggesting that their contribution to paradoxical secretion should be very limited. Somatogonadotropes were present only in females and contained adrenocorticotropic hormone. They responded to growth hormone-releasing hormone but failed to respond to gonadotropin-releasing hormone (LHRH). Other polyhormonal cells identified include (1) gonadocorticotropes, restricted to females, where they make up more than 50% of all the gonadotropes and contain other AP hormones; (2) gonadomammotropes, which are present preferentially in female cells and respond to LHRH; and (3) gonadothyrotropes, which are present similarly in male and female pituitaries.