A combinatorial RNA library approach, denominated SELEX technique (Systematic Evolution of Ligands by Exponential Enrichment), was employed to identify subtype-specific inhibitors of purinergic receptor P2X₂ and P2X₄ subtypes in order to study the functions of these receptors during neuronal differentiation of P19 embryonal carcinoma cells in vitro. The evaluation of exact physiological functions of metabotropic (P2Y) and ionotropic (P2X) receptor subtypes was hindered in the past by the missing of subtype-specific agonists and antagonists. We have performed reiterative SELEX cycles using membrane fractions from 1321N1 glial cells expressing recombinant P2X₂ or P2X₄ receptors as selection targets. Following 8 SELEX cycles the selected aptamers inhibited either P2X₂ or P2X₄ receptor activation, as determined by whole-cell recording in the presence of ATP. Aptamers selected against P2X₂ receptors did not affect the activity of the P2X₄ receptor, and P2X₄ receptor-specific aptamers did not inhibit P2X₂ receptor function. This work describes for the first time the identification of inhibitors which can differentiate between P2X₂ and P2X₄ receptor subtypes.