The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap4A and Ap5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap4A content per mg of protein was 169 +/- 25 pmol and 159 +/- 22 pmol for Ap5A. The study of the exocytotic release of these compounds was carried out with 100 microM 4-aminopyridine or 10 microM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 +/- 3.0 pmol/mg protein of Ap4A and 11.6 +/- 2.4 pmol/mg protein of Ap5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 +/- 3.0 and 16.6 +/- 2.8 pmol/mg of protein of Ap4A and Ap5A respectively. The ratios of exocytosis were close to 7-9% and 10-12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.