The distribution patterns of selected calcareous nannofossils were investigated by means of quantitative methods in the middle Miocene stratigraphic record of the Mediterranean region. Our goal was to test the reliability of the biohorizons used in various zonations. The biostratigraphic reliability has been evaluated by considering the mode of occurrence of the biohorizons and by their potential of correlation among distant and various facies sections. Most biohorizons used in the standard zonations are of limited utility in the region, because some marker species are missing (Catinaster coalitus, Catinaster calyculus) or poorly represented (Discoaster kugleri). By utilizing distinct changes in the distribution pattern of forms common in the region, like first common and continuous occurrence (FCO), last common and continuous occurrence (LCO), absence (paracme) intervals, besides classical first occurrence (FO) and last occurrence (LO) datums, at least 12-13 biohorizons can be consistently correlated among the considered sections. On the basis of these, we established a set of ten regional biostratigraphic zones and subzones, which can be recognized in various facies sediments. The average time resolution of the proposed zonal system is about 1 m.y., which can be substantially improved by integrating calcareous nannofossil and planktonic foraminifer biostratigraphies. The study of the stratotype sections of the Langhian and Serravallian stages, indicates that the boundary between the two units is approximated by the LO of Sphenolithus heteromorphus, and is not associated with the FO of Discoaster kugleri as assumed previously. The base of the Langhian (lower Miocene/middle Miocene boundary) does not correspond to a calcareous nannofossil zonal boundary; it occurs in the upper part of Zone NN4 (CN3). The state of the Serravallian-Tortonian boundary (middle Miocene/upper Miocene boundary) is discussed. The proposal of defining the boundary by the FO of Neogloboquadrina acostaensis is supported. This biohorizon occurs close to the LO of Helicosphaera walbersdorfensis in the Mediterranean region and close to the FO of Catinaster coalitus in many oceanic area