Introduction Neurokinin B (NKB), dynorphin A (Dyn A) and kisspeptin (KiSS) are key agents that participate in the neuroendocrine regulation of the development and functioning of the reproductive system. While the role of KiSS is better understood, the functions of NKB and Dyn A at the pituitary level have not been elucidated. The objective of our study was to analyse their direct effect on kisspeptin-10 (KiSS-10) secretion by anterior pituitary cells isolated from pubescent ewes. Material and Methods Pituitary cells from 10-month-old ewe lambs were incubated in McCoy’s 5A medium without hormones (the control), or with 10−11, 10−10, 10−9, 10−8 or 10−7 M of NKB or Dyn A for 2, 4, 6, 12, 18 or 24 h. The concentration of KiSS-10 was analysed by ELISA using species-specific antibodies. Results When applied at the concentrations of 10−10–10−7 M, NKB increased KiSS-10 secretion throughout the entire experiment (2–24 h), compared to the control. Significantly higher (P-value ≤ 0.05) KiSS-10 release than in the control was observed after 6–24 h exposure of the cells to 10−8 M of NKB. However, no effect of NKB on the secretion of KiSS-10 was shown when applied at the lowest concentration (10−11 M). In turn, there was no significant effect of Dyn A at any concentration on KiSS-10 secretion by pituitary cells at any time Conclusion In contrast to Dyn A, NKB can directly affect KiSS-10 secretion from the pituitary cells of pubescent ewes in a way dependent on the time of exposure to this neuropeptide and its concentration in the culture medium. This phenomenon may indicate a potential role of NKB in the initiation of reproductive activity, which leads to the achievement of sexual maturity in the optimal developmental window.