Biochemical studies demonstrate that three steroids postulated to function as the sea lamprey migratory pheromone are released in sufficient quantities, and possess adequate stability and binding characteristics, to function as a multi-component pheromone in natural river waters. Mass spectrometric (MS) analyses of the holding water of recently fed larval lamprey demonstrated that each of these compounds is released at rates of 5-25 ng larva(-1) h(-1), adequate to produce picomolar (biologically relevant) concentrations in river waters. Petromyzonamine disulfate (PSDS) was released at about twice the rate of the other two components, petromyzonamine disulfate (PADS) and petromyzonol sulfate (PS). Unfed larvae also released all three steroids but only at about two-thirds the rate of fed larvae and in a different ratio. However, a behavioral test of fed and unfed larval holding waters suggested this change in pheromone ratio does not diminish pheromonal signal function in the winter when larvae are not feeding. A study of steroid degradation found that PADS and PSDS had half-lives of about 3 days, similar to values previously described for PS and sufficiently slow for the entire pheromone to persist in river mouths. Finally, both MS and electro-olfactogram recording found that contrary to previous suggestions, natural levels of natural organic matter found in streams do not bind to these steroids in ways that diminish their natural biological potency. In conclusion, it appears highly likely that a mixture of PADS, PSDS and PS is present at biologically relevant concentrations and ratios in many Great Lakes streams where it functions as a pheromonal attractant.