Pregnant women at risk of premature labor receive a single treatment with the synthetic glucocorticoid dexamethasone (DEX) between the 24th and 34th week of pregnancy to ensure adequate maturation of the fetal organs. Due to the postponement of the due date, several doses are often administered. Because purinergic signaling plays an important role in the development of the auditory brainstem, particularly the cochlear nucleus, we investigated whether repeated DEX treatments affect components of the ADP-mediated (NTPDase2 and P2Y1R) and adenosinergic (CD73 and A1R) signaling systems. To compare the effects of repeated and single DEX treatment, pregnant C57BL/6 dams received DEX (0.4 mg/kg) on gestation days 15-17 for repeated treatment and 15 for single treatment followed by saline for two days. Control group received saline. Auditory brainstem tissue was harvested from the pups at postnatal days 8, 14 and 20 (PD8, 14, 20). The mRNA content was analyzed by qRT-PCR, while tissue distribution and cellular localization were examined by double immunofluorescence using VGLUT1 as a marker for glutamate synapses. All analyzed components of the purinergic signaling system were expressed in the cochlear nucleus of PD8-20. NTPDase2, P2Y1R and A1R were associated with or in close proximity to neuronal cells, while CD73 immunoreactivity was pronounced in vascular elements. Both single and repeated treatment resulted in a decrease in P2Y1R expression in PD8, while NTPDase2 expression was altered in PD20 in both treatment groups. We conclude that repeated prenatal DEX treatment could affect the ADP signaling system, but further studies should be conducted.