Red-winged Blackbirds (Agelaius phoeniceus) are a frequently parasitized host species of obligate brood parasitic Brown-headed Cowbirds (Molothrus ater). Yet, this common host does not reject parasite eggs or young. A hypothesis to explain the lack of cowbird egg and nestling rejection is that redwings frontload their responses by investing into antiparasitic nest defense behaviors toward female cowbirds, relative to non-laying male cowbirds and non-threatening other species. A review of prior studies using cowbird-mount presentations with or without acoustic playbacks supported some but not all predictions of this hypothesis. We conducted a new study at two geographically separate sites, in New York City, NY and in Ithaca, NY, USA, where Red-winged Blackbirds were presented with taxidermic models on a tripod and species- and sex-specific vocalization playbacks of female or male Brown-headed Cowbirds, female Northern Cardinals (Cardinalis cardinalis), or a tripod and silence (control). Reactions of both sexes to mount and playback presentations near known, active nests, conducted repeatedly throughout the breeding cycle, were supplemented with mount and playback presentations to males without known nests. Red-winged Blackbirds responded more aggressively toward presentations of both sexes of the parasite than to the cardinal and the tripod treatments. In addition, they responded more aggressively at known nests to mounts presented closer rather than farther, and at nests with nestlings rather than eggs. Furthermore, aggressive reactions were more frequent in Ithaca, NY than in New York City, NY. Finally, the reactions of female and male redwings near known active nests and of males without known nests showed statistically similar patterns, implying that data from these two types of experimental contexts could be combined in our analyses, and perhaps in future studies, to more efficiently characterize this host's responses to parasite presentations.