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 B 1 , B 2 and B 3 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.