Dictyostelium mutants expressing aequorin were used to study and compare the roles of heterotrimeric G-proteins and the second messengers IP3 and cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals. The calcium responses of vegetative cells to folate were dramatically impaired in Gbeta and Galpha4 null mutants but were restored with altered kinetics and temperature-sensitivity in Gbeta null mutants overexpressing wild type and temperature-sensitive Gbeta isoforms. Folic acid receptors thus mediate changes in [Ca2+]i via a Galpha4betagamma-dependent pathway. Neither folate nor cAMP-induced [Ca2+]i signals were significantly altered in PLC null transformants, but [Ca2+]i changes elicited by both attractants were significantly prolonged in two stmF mutants lacking cGMP-specific phosphodiesterase activity. This confirms an important role of cGMP in regulating receptor-activated Ca2+ uptake and/or extrusion systems. This cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally down-regulated and all but disappeared by the time the cells reached full aggregation competence after 8 h of starvation. The results suggest that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a complex manner via multiple signalling pathways, one that is G-protein- and cGMP-dependent (present at the vegetative and early poststarvation stage) and another that is G-protein-independent (dominant in fully aggregation-competent cells at approximately 8 h poststarvation).