The aim of the study was to determine an in vitro effect of specific agonists of opioid receptors on basal prolactin secretion and in the presence of dopamine or thyreoliberin (TRH) by porcine anterior pituitary cells. The cells were isolated from anterior pituitaries of gilts on days 8–10, 15–17 and 19–21 of the oestrous cycle and submitted to in vitro culture with mu-, delta- and kappa opioid receptor agonists – FK 33-824, DPLPE and U 50,488, respectively. Differentiated effects of the opioid agonists on prolactin secretion by isolated pituitary cells of gilts in chosen days of the oestrous cycle were shown. In the midluteal phase (days 8–10), a reduced prolactin secretion was demonstrated after activation of mu-, delta- and kappa-opioid receptors under all tested conditions. In the early follicular phase (days 15–17), the activation of mu-, delta- and kappa-opioid receptors increased prolactin secretion under basal conditions, as well as mu- and delta-opioid receptors – in the presence of TRH, but the stimulation of mu- and kappa-opioid receptors reduced the hormone secretion in the presence of dopamine. In the late follicular phase (days 19–21), kappa-opioid receptor agonist stimulated prolactin secretion under all tested conditions. The activation of mu- and delta-opioid receptors increased prolactin secretion under basal conditions and in the presence of dopamine, but decreased – in the presence of TRH. The results suggest a possibility of diverse participation of endogenous opioids, depending on stage of the oestrous cycle, in the modulation of prolactin secretion at the pituitary level in gilts during the oestrous cycle.