In vitro cultivation of adult digeneans can benefit research on their biology and contribute to the development of new drugs and vaccines. Successful in vitro growth of excysted metacercariae into adults capable of producing embryonated eggs typically requires that the worms be inseminated. The goal of the study was to develop an in vitro insemination procedure for the progenetic microphallid digenean Gynaecotyla adunca. To do so, we determined the length of time needed for in vitro sperm development in excysted metacercariae and whether the adult worms could self-inseminate in the absence of conspecifics. We also examined the effect of different culture vessels, worm densities, incubation temperatures, length of time incubated with conspecifics, and different pH levels on the percentage of worms inseminated. We found that sperm maturation time for G. adunca was 8–10 hr postexcystment. In the absence of conspecifics, the parasite did not self-inseminate. We observed the highest percentage of inseminated worms when 50 excysted metacercariae were incubated at 37 C for 48 hr in 15-ml conical-bottom tubes containing pH 7 Hank's balanced salt solution. Furthermore, freshly excysted worms incubated in these conditions and then transferred to culture in Dulbecco's modified Eagle medium/F-12 medium and horse serum deposited normal-shaped, embryonated eggs. Our findings provide the basis for a straightforward, reproducible procedure that permits the in vitro insemination of the parasite G. adunca and should be applicable to other progenetic digeneans.