In Chile, the horn fly, Hematobia irritans (L., 1758), is a major pest of grazing cattle and affects livestock production during the summer. Previous studies in Europe and the United States have shown that cattle flies, including H. irritans, are differentially attracted to individual cattle within herds and that volatile semiochemicals are responsible for this phenomenon. This study provides evidence that similar differential attractiveness occurs for the interaction between Chilean Holstein-Friesian cattle herds and local H. irritans populations. Thus, Holstein-Friesian dairy cattle, Bos taurus, which were of similar age and physiological condition, were shown to possess an uneven distribution of H. irritans. Heifers h6904 and h8104 were defined as low-carrier heifers and h5804, h2304 and h1404 as high-carrier heifers. Gas chromatography (GC) and coupled GC-mass spectrometric (GC-MS) analysis of samples collected from heifers revealed the presence of compounds previously reported as semiochemicals for cattle flies, including meta- and para-cresol, methylketones (C8–C11), and 6-methyl-5-hepten-2-one. Other compounds identified included carboxylic acids (butanoic, 3-methylbutanoic, pentanoic, and hexanoic acids), 1-hexanol, and 3-octanone. In Y-tube olfactometer studies, both m- and p-cresol attracted H. irritans at the highest doses tested (10-6 g), as did the positive control l-octen-3-ol. Of the other compounds tested, only 2-decanone and 2-undecanone produced a behavioral response, with significantly more flies being recorded in the control arm when the former compound was tested (at 10^-6 and 10^-8 g), and more flies being recorded in the treated arm for the latter compound (at 10^-7 g). This demonstration of behavioral activity with the identified compounds represents a first step for research into the application of semiochemicals in monitoring and control of cattle flies in Chile.