1. Diadenosine tetraphosphate (Ap4A) a dinucleotide stored and released from rat brain synaptic terminals presents two types of affinity binding sites in synaptosomes. When [3H]-Ap4A was used for binding studies a Kd value of 0.10 +/- 0.014 nM and a Bmax value of 16.6 +/- 1.2 fmol mg-1 protein were obtained for the high affinity binding site from the Scatchard analysis. The second binding site, obtained by displacement studies, showed a Ki value of 0.57 +/- 0.09 microM. 2. Displacement of [3H]-Ap4A by non-labelled Ap4A and P2-purinoceptor ligands showed a displacement order of Ap4A > adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S) > 5'-adenylyl-imidodiphosphate (AMP-PNP) > alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) in both sites revealed by the Ki values of 0.017 nM, 0.030 nM, 0.058 nM and 0.147 nM respectively for the high affinity binding site and values of 0.57 microM, 0.87 microM, 2.20 microM and 4.28 microM respectively for the second binding site. 3. Studies of the P2-purinoceptors present in synaptosomes were also performed with [35S]-ADP-beta-S. This radioligand showed two binding sites the first with Kd and Bmax values of 0.11 +/- 0.022 nM and 3.9 +/- 2.1 fmol mg-1 of protein respectively for the high affinity binding site obtained from the Scatchard plot. The second binding site showed a Ki of 0.018 +/- 0.0035 microM obtained from displacement curves. 4. Competition studies with diadenosine polyphosphates of [35S]-ADP-beta-S binding showed a displacement order of Ap4A > Ap5A > Ap6A in the high affinity binding site and Ki values of 0.023 nM, 0.081 nM and 5.72 nM respectively. The second binding site potency order was Ap5A> Ap4A > Ap6A,with Ki values of 0.28 microM, 0.53 microM and 5.32 microM respectively.5. Displacement studies of [35S]-ADP-beta-S with P2-purinoceptor agonists showed the following potency pattern: ADP-beta-S > AMP-PNP >alpha,beta-MeATP with Ki values of 0.021 nM, 0.029 nM 0.215 nM respectively in the high affinity binding site. 2-Methylthio-adenosine 5'-triphosphate (2MeSATP) was unable to displace [35S]-ADP-beta-S in this binding site. The second binding site showed a profile of ADP-beta-S> a,beta-MeATP> AMP-PNP > 2MeSATP and Ki values of 0.0 18 microM, 0.212 microM, 0.481 microM and 18.04 microM respectively.6. These studies suggest the presence of a new P2-purinoceptor in rat brain synaptosomes with high affinity for diadenosine polyphosphates which we tentatively designate as P2d.