Summary Canine dirofilariasis is widespread in urban areas of central and northern Colombia. Previously we detected specific antibodies against complex antigens from Dirofilaria immitis adult worms in individuals from an isolated Tikuna Indian community in the Colombian Amazon. In this study a 56 kDa polypeptide from the adult D. immitis excretory/secretory (E/S) products is identified by Western blot, isolated by elution from polyacrilamide gels and applied in an ELISA‐based test for the detection of specific IgG. Eleven of 74 serum samples analysed were positive by ELISADi56. Positive individuals came from five different areas of Colombia. The highest number of positives was found in the Amazon (4), followed by Bogotà (3). The physicians of the area must be alerted regarding the existence of human D. immitis infections and include dirofilariasis in the differential diagnosis of pulmonary nodules.