Phyllosticta ampelicida conidia germinate only after making contact with and attaching to a substratum. Previous studies suggested a role for Ca2+ in this process. A Ca2+ buffering system was used to control the external free Ca2+ concentration. Both germination and appressorium formation were reduced or abolished with low Ca2+ (less than or equal to nanomolar levels) but were nearly 100% at millimolar levels of Ca2+. Germination initiation required Ca2+ within 10-25 min after the spore made contact with the substratum. Appressorium initiation required Ca2+ 90-120 min following initial contact. Ca2+ channel blockers nicardipine and lanthanum abated spore development. TMB-8, a blocker of internal Ca2+ channels, reduced both developmental events. Gadolinium, a putative stretch-activated Ca2+ channel blocker, abolished both developmental events at nanomolar levels. Calmodulin antagonists, compounds R-24751 and 48/80, abated spore development at micromolar levels. Together, these results suggest that Ca2+ signaling is involved in both germination and appressorium formation in P. ampelicida pycnidiospores.