The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. Isolations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 mu m, and a maximum length of ca. 5 mu m. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.