We performed phylogenetic analyses of the subfamily Rubioideae (Rubiaceae) based on three different pieces of chloroplast DNA, the protein codingrbcL gene, the spacer sequence betweenatpB andrbcL (atpB-rbcL), and the recently published (Andersson and Rova 1999)rps16 intron data. NewrbcL sequences have been produced for 41 taxa and there are 52 newatpB-rbcL spacer sequences. All analyses gave similar results concerning the phylogeny, but they differ slightly in resolution and support for the various branches. The minor tribes Ophiorrhizeae, Urophylleae, Lasiantheae, and Coussareeae form a grade to the rest of the subfamily, which consists of two well-supported branches, the Psychotrieae alliance and the Spermacoceae alliance, including a majority of all genera and species. Based on the resulting phylogenies we present a revised classification of the Rubioideae. We accept 16 tribes of which 12 more or less correspond to earlier tribal circumscriptions: Anthospermeae, Argostemmateae, Craterispermeae, Gaertnereae, Morindeae, Paederieae, Psychotrieae, Schradereae, Spermacoceae, Rubieae, Theligoneae, and Urophylleae; two tribes have received new and very different circumscriptions: Ophiorrhizeae and Coussareeae; and two are new to science: Lasiantheae and Danaideae.