Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.