In August 2008, West Nile disease re-emerged in Italy. The infection is affecting the North Eastern regions and, as of November 2008, has caused 33 clinical cases and five fatalities in horses. Until now, no deaths have been reported in birds. Mosquitoes, blood, serum and tissue samples, from horses and birds, within and around the outbreak area, have been collected and tested by various methods both serologically and virologically. West Nile virus strains have been isolated from blood samples of one horse and one donkey and from pools of brain, kidneys, heart and spleen of a pigeon and three magpies. When compared to the strain isolated during the 1998 Tuscany outbreak, the 255 bp sequence of the genome region coding for the envelope (E) protein of the isolated WNV strains, exhibited a 98.8% and 100% similarity at nucleotide and amino-acid level respectively.