Background: 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. Objective and Methods: 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. Results: 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. Conclusion: 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.