We investigated the variation of wood anatomical characteristics and carbon isotopic composition of tree rings showing intra-annual density fluctuations (IADFs) in plants of Pinus pinaster Ait. growing at a coastal plantation in Tuscany (Italy). IADFs are regions of:the tree ring Where wood density changes abruptly due to a sudden change of environmental conditions, particularly of water availability. Dendrochronological analyses allowed dating of the rings and four regions were considered in each tree ring: earlywood, IADF, late-earlywood and latewood. Although IADF commonly has been classified as latewood-like tissue in the literature, we found differences in anatomical characteristics and carbon isotopic composition between tracheids of the two regions. The lumen area of tracheids in IADF was significantly larger than in latewood, while still smaller than in earlywood and late-earlywood. Latewood and IADF had a greater proportion of narrow tracheids than both earlywood and late-earlywood. Although latewood and IADF were characterized by tracheids with lumina lengthened in the tangential direction, while earlywood tracheids were elongated in the radial direction, some differences were found also between latewood and IADF. Moreover, IADF tracheids had a higher ¹³C/¹²C ratio than any other region and showed isotopic values significantly different from the latewood. The quantification of anatomical features of tracheids within rings was useful to discriminate between latewood and IADFs, as well as helpful for the identification of tree-ring boundaries. The overall interpretation of dendrochronological, wood anatomical and carbon isotopic data seems to be a promising approach for the dating and the ecological interpretation of tree rings in Mediterranean ecosystems and for gaining climatic information with intra-annual resolution.