The hydrophobic surfaces presumably involved in the membrane interaction of pertussis toxin have been mapped by a new detergent-binding assay. This is based on the interdispersion among detergent micelles of trace amounts of radioactive photoreactive phospholipid analogues, able to cross-link to the protein thereby labelling its detergent-binding domains. The assay has proven to be very sensitive. Subunits B1, B2 and B3 of pertussis toxin were found to interact with the lipid micelles suggesting that they may be involved in the membrane penetration step of the intoxication process.