The effect of blood-feeding on total and specific immunoreactive ecdysteroids in Stomoxys calcitrans adult females was examined following the fourth and fifth blood meals when total whole body and hemolymph ecdysteroids showed a dramatic increase in the titer. In general, for both total and specific immunoreactive ecdysteroids that included highly polar material, 20,26-dihydroxyecdysone, 20-hydroxyecdysone and ecdysone, there were clear differences between the effects of the fourth and fifth meals. Following the fifth meal, the titers rose sooner, reached higher levels and remained high longer than those following the fourth meal. This is the first examination of the effects of back-to-back blood meals on total and specific ecdysteroid levels in an intermittent, blood-feeding fly. These results suggest that both rates of synthesis and degradation are affected by blood-feeding and that the number and possibly quantity of blood ingested affect the biochemical mechanisms that regulate ecdysteroid titers in S. calcitrans.