Formica polyctena antidiuretic factor (FopADF) was purified from a 15% trifluoroacetic acid (TFA) extract of the abdomens of 150,000 worker ants. After solid phase extraction of the crude extract and reversed-phase HPLC on two C(18) columns, an antidiuretic factor was isolated. Tested at a concentration of 1.0 ant-equivalents/µl (ant-eq/µl), the factor reversibly inhibited fluid secretion of isolated Malpighian tubules to 29+/-5% (mean+/-SE, n=24) of the control value. The same concentration of FopADF reversibly depolarized both the basolateral membrane potential (V(bl)), from -21+/-2 mV to -3+/-1 mV (n=5), and the apical membrane potential (V(ap)), from -65+/-5 mV to -20+/-5 mV (n=5). Similar effects on fluid secretion and V(ap) were caused by a TFA extract of the haemolymph of ants with non-secreting tubules. Unfortunately, further purification of FopADF on a C(4) column led to a loss of activity in the fluid secretion assay. This is the first time an endogenous antidiuretic factor acting directly on Malpighian tubules has been partially purified and shown to depolarize the tubule cell membranes.