Understanding signaling pathways that modulate conidiation of mitosporic fungi is of both practical and theoretical importance. The enzymatic origin of nitric oxide (NO) and its roles in conidiation by the sclerotial parasite Coniothyrium minitans were investigated. The activity of a nitric oxide synthase-like (NOS-like) enzyme was detected in C. minitans as evidenced by the conversion of l-arginine to l-citrulline. Guanylate cyclase (GC) activity was also detected indirectly in C. minitans with the GC-specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which significantly reduced production of cyclic GMP (cGMP). The dynamics of NOS activity were closely mirrored by the cGMP levels during pycnidial development, with the highest levels of both occurring at the pycnidial initiation stage of C. minitans. Furthermore, the NO donor, sodium nitroprusside (SNP), stimulated the accumulation of cGMP almost instantly in mycelium during the hyphal growth stage. When the activity of NOS or GC was inhibited with Nomega-nitro-l-arginine or ODQ, conidial production of C. minitans was suppressed or completely eliminated; however, the suppression of conidiation by ODQ could be reversed by exogenous cGMP. The results also showed that conidiation of an l-arginine auxotroph could be restored by the NO donor SNP, but not by cGMP. Thus, NO-mediated conidiation has more than one signal pathway, including the cGMP signal pathway and another yet-unknown pathway, and both are essential for conidiation in C. minitans.