Strains of the Gram-negative bacterium Klebsiella pneumoniae secrete pullulanase, a maltose-inducible starch debranching enzyme that exists as a cell surface bound intermediate. Three classes of secretion mutants were obtained by transposon In 10 mutagenesis. Class I and class III mutants carry Tn10 insertions in pullulanase secretion genes. Class II mutants carry insertions in regulatory loci that are required for the high level expression of pullulanase and other maltose-inducible genes (the maltose regulon). One such locus appears to correspond to a previously unknown locus. The phenotypes of the secretion mutants and the analysis of E.coli expressing pullulanase and/or cloned pullulanase secretion genes suggest that pullulanase secretion functions are involved in translocating pullulanase across the outer membrane and in releasing it from the cell surface. Most if not all pullulanase secretion genes are located to both sides of the structural gene for pullulanase (pu1A). Pullulanase was found to be a lipoprotein. Surprisingly, secreted pullulanase also carried lipid. However, strain K21 differed from other strains of Klebsiella pneumoniae by its ability to secrete not only acylated pullulanase but also a second, unacylated form of pullulanase. Strain K21 is also unusual because of its ability to secrete most pullulanase during logarithmic growth. This pullulanase corresponds to the unacylated pullulanase, with the remaining secreted pullulanase being acylated and secreted during stationary phase, as is the case for most or all pullulanase of other strains of Klebsiella pneumoniae. Strain K21 is also unusual because of the high level expression of the maltose regulon, including pulA, in the absence of maltose. This property, including the unusual features described above, may be a consequence of the selection of strain K21 for high level commercial production of pullulanase. Models for pullulanase secretion are discussed and approaches towards increasing the efficiency of commercial pullulanase production by strain K21 are outlined.