Background:Lipases (acylglycerol acylhydrolase, E. C. 3. 1. 1. 3) are widely distributed among microorganisms, animals and plants, catalyzing the hydrolysis of triglycerides to free fatty acids and glycerol. Their commercial application includes pharmaceutical, chemical, and paper industries.Objectives:This study aimed to isolate and screen lipolytic fungi from coastal waters of the southern Caspian Sea by Internal Transcribed Spacer-Polymerase Chain Reaction (ITS-PCR), and to optimize their lipolytic activity, pH and temperature. The ITS regions possess a high variation among taxonomically distinct fungal species and even within species.Materials and Methods:All fungal were tested to determine their lipolytic activity by the Tributyrin agar plate assay. After DNA extraction, lipase-producing fungi were identified via ITS-PCR of rDNA region with ITS1 and ITS4 primers.Results:Four fungal species were isolated from water samples of the Caspian Sea (north of Iran) between February and June 2011. The nucleotide sequences reported for three of these isolates have been assigned accession numbers from NCBI Gene Bank database. Among these species, Cladosporium langeronii showed maximum lipolytic activity (34 U/mL) and maximum clear zone formation (6 mm) on the Tributyrin agar plates. The optimum pH and temperature for activity were 8.0 and 35°C, respectively.Conclusions:The findings of this study indicated that these isolates were plant pathogenic fungi, which entered seawater from the environment, and were likely to have a suitable lipase activity on plant oils.