Phosphorylated mitogen-activated protein kinases (MAPK) transmit signals by activation of their targets. The extent of signal transduction could depend on MAPK phosphorylation level, concentration, and subcellular localization. The pathogenicity MAPK Chk1 of the fungal corn pathogen Cochliobolus heterostrophus is required for central developmental functions, including appressoria formation, conidiation, melanization, virulence, and female fertility. We followed CHK1 transcript level, protein localization, quantity, phosphorylation, and expression of downstream genes during conidial germination on a surface inductive for appressoria formation and in suspension. The Chk1-GFP protein representing a translational fusion of Chk1 and GFP (green fluorescent protein) was very abundant in ungerminated conidia, accumulated in maturating appressoria and appressorial nuclei, but was uniformly distributed in suspension-grown hyphae. Expression of Chk1-dependent genes was upregulated in appressoria-forming hyphae but not in suspension. Despite Chk1 activation, there was no change in its phosphorylation and total protein quantity. Of all conditions tested, a temperature shift caused a decrease whereas hyperosmotic stress caused an increase in Chk1 phosphorylation. Activation of Chk1 during appressoria formation is apparently manifested by its local accumulation but not by significant changes in phosphorylation.