The 3' untranslated region (3' UTR) of the bovine natural resistance-associated macrophage gene (NRAMP1 or SLC11A1) was genotyped in Colombian Creole Blanco Orejinegro (BON) (Bos taurus) (n = 140) and Zebu Brahman (Bos indicus) (Z) (n = 20) cattle and their crosses (BON x Zebu Brahman [B x Z] [n = 10]; Zebu Brahman x BON [Z x B] [n = 10]), and in animals from a Holstein x BON (H x B) (n = 10) cross. Direct sequencing and single-strand conformation polymorphism analysis (SSCP) helped in detecting the polymorphic behaviour. The association between resistance to brucellosis infection and SSCP genotype was evaluated using a macrophage in vitro killing assay employing a virulent Brucella abortus strain. The 3' UTR (GT) repeated polymorphism was gentoyped and its association with resistance to brucellosis was evaluated. When all breeds were grouped, a high frequency in the homozygote GT(12) (AA genotype) (0.823) and a very low frequency in the homozygote GT(10) (BB genotype) (0.047) were detected. The BON (0.963), Z x B (0.60) and H x B (1.00) cattle showed high GT(12) allele frequencies, unlike that seen for the B x Z and Zebu cattle (0.3002 and 0.218, respectively). The GT(10) allele was only found in the Zebu cattle (0.391). A significant association (p < 0.001) was found between the B. abortus macrophage in vitro killing assay phenotypes and the bovine SLC11A1 3' UTR genotypes, which suggests that the A allele may be associated with resistance. Because only nine animals had the BB genotype, the results require some confirmation in more extensive populations.