Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichum-gloeosporioides. As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased. Nitrogen assimilation also was required for PL secretion at pH 6.0. Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0. Sequence analysis of the 5′ upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC. Consistent with this result, the transcript levels of pac1 (the C. gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH. Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C. gloeosporioides.