A single dose (1 mg/kg) of milbemycin D was administered orally to 24 dogs with microfilaremia of Dirofilaria immitis, and the number of circulating microfilariae was counted weekly. The number was decreased by 3 to 8% of the pretreatment levels 1 week after the drug administration. The number remained relatively stable for the first 8 weeks and was gradually increased thereafter without returning to the pretreatment levels by 20 weeks. Three or 4 dogs each were euthanatized on day 1, and 1, 4, 8, 12, 16 and 20 weeks after the drug administration to examine the effects of the drug on intrauterine microfilariae and embryos of the worms. Although no intrauterine microfilariae were destroyed directly by the drug, degeneration and collapse of morular embryos and decrease in the number of intrauterine microfilariae were observed 12 after weeks the drug administration. These findings became more remarkable with time, and no intrauterine microfilariae developed in any worms by 20 weeks. The electron-microscopic findings revealed that the nucleoli of oocytes had a high density in the worms of 1, 4 and 8 week groups. Unequal size of cleavage cells and decrease of polysome number were noticed in the early-stage embryos after 8 weeks. It was assumed that the drug might have some effect on the chromosomes or genes in the germinal stem-cell of the heartworm and interfere with protein syntheses, resulting in inhibition of embryonic development. Twelve dogs were given milbemycin D (1 mg/kg) a total of 4 or 6 times monthly according to a prophylactic program.(ABSTRACT TRUNCATED AT 250 WORDS)