A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.