Background and Objective: Cephalosporin acylases (CA) are highly specialized peptidases that are able to break down the amide bond between the lactam nucleus and lateral chain without destruction of beta-lactam ring. This enzyme transmutes the cephalosporin C to 7-ACA. The 7-ACA is an important intermediate in synthesizing the semi-synthetic cephalosporin in pharmaceutical industries. The purpose of this study was to screen and optimize the species having maximum amount of production of cephalosporin acylase enzyme isolated from clinical samples among some gram- negative bacteria producing cephalosporin acylase. Materials and Methods: Out of a total of 60 species gathered from several hospitals in different places of Tehran city, the clinical species such as Pseudomonas, Acintobacter and Escherichia coli were isolated. At first, the resistant species were differentiated by antibiogram method. Then, the activity of enzyme was measured by spectrophotometer. After identification and isolation of the enzyme producing species, the effect of factors such as time, temperature, pH and the amount of aeration of cultivation environment on the amount of enzyme production was investigated. Results: In this study, among the three groups of bacteria mentioned above, a species of Pseudomonas aeroginosa showed a significant cephalosporin acylases activity. Optimum activity for this species regarding the mentioned factors was estimated as follows: pH 8, incubation time 48 h, temperature 37 C and the component of aeration was 150 rpm. Conclusion: The findings point out that Pseudomonas species has a significant cephalosporin acylases activity compared with other species examined in this study. Therefore, this species may have implication for industrial production of the above mentioned enzyme and also can be used in related researches.