Acinetobacter spp. are responsible for an increasing number of opportunistic, nosocomial infections. They have been isolated from diverse inanimate objects in the hospital environment and are resistant to most of the commonly used antibiotics. Existing media for the isolation of Acinetobacter spp. are either nonselective, allowing the growth of unwanted bacteria, or too inhibitory, inhibiting the growth of many Acinetobacter strains. For the rapid isolation and effective control of Acinetobacter infection, a new selective and differential medium, Leeds Acinetobacter Medium (LAM), has been developed to isolate Acinetobacter spp. from clinical and environmental sources. The concentration of antibiotics and other ingredients in this medium have been determined according to the results of MIC and viable counts performed for these ingredients. LAM was compared with other selective and differential media for the isolation of Acinetobacter spp. from a local hospital environment and proved to be better in terms of recovery and selectivity.