We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hyperscretion of phospholipase A(1) (PLA(1)). Using random chemical mutagenesis,. uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA(1)-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA(1) activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA(1); no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA(1) in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA(1) is not available on the commercial market for fine chemicals and little is known about the role of PLA(1) in cell signaling and metabolism.