Abstract: Medically useful semisynthetic cephalosporin antibiotics are made from precursor 7-aminocepha-losporanic acid (7-ACA). Cephalosporin Acylase (CA), which catalyzes hydrolysis of both glutary l-7-aminocephalosporanic acid (GL-7ACA) and cephalosporin C (CPC) to 7-ACA, is thus a very important enzyme for producing semisynthetic beta-lactam antibiotics. The cephalosporin acylase can be found in several Pseudomonas sp. (such as P. putida, P. cepacia BY 21 P. nitroreducens, P. syringae, p. SE83, p. V22, p. SY-77, p. sp.130 ,�) and other bacteria. Therefore, screening of cephalosporin asylase positive pseudomonas is very important. We isolated 130 Pseudomonas sp. from clinical and environmental specimens (75 clinical samples, 40 hospital environment samples, 10 water samples, 5 soil samples). All of isolated Pseudomonas sp. were tested for cephalosporin acylase by P.C.R method and analysis of gel electrophoresis patterns. We found only 2 cephalosporin acylase pseudomonas positive from clinical samples. These strains were selected for sequencing and cloning in E. coli and assessment of gene expression and cephalosporin acylase production in others stages of our study.