<b>Background:</b> In tropical climates, sensitization to Bloma tropicalis and Dermatophagoides pteronyssinus is high and mainly directed to species–specific allergens. There is some cross–reactivity between extracts of these mites, probably due to the group 5 allergens that have high sequence homology. <b>Objective and Methods:</b> We used the radioallergosorbent test (RAST), RAST inhibition and immunoblotting inhibition experiments to investigate the cross–reactivity between the recombinant allergens BtM and Der p 5, expressed as glutathione S–transferase fusion proteins, to detect the epitopes involved and to analyze the importance of this cross–reactivity. <b>Results:</b> Seventy–nine percent of 48 patients sera were RAST positive to both recombinants, with a strong correlation (r = 0.8, p<0.0001). BtM inhibited 25 and 21.1% of IgE–binding to B. tropicalis and D. pteronyssinus extracts respectively and Der p 5 inhibited 22 and 24% of IgE–binding to D. pteronyssinus and B. tropicalis extracts. Furthermore, BtM inhibited 74.5% of IgE binding to Der p 5 and Der p 5 inhibited 72.4% of IgE–binding to BtM. RAST inhibition with BtM–derived synthetic peptides showed that peptide 4 (residues 35–50) and peptide 5 (residues 46–61) inhibited 37 and 16% of IgE–binding to BtM while peptides 5 and 2 (residues 14–30) were able to inhibit the IgE binding (32 and 28%, respectively) to Der p 5. <b>Conclusion:</b> There is cross–reactivity between BtM and Der p 5, which explains almost all the cross–reactivity between the two mite extracts. This cross–reactivity seems to be related to epitope(s) at the C–terminal segment of these allergens.