The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-11, and Er-20, secreted by cells of different mating types of the ciliated protozoa Euplotes raikovi, have been determined by automated Edman analyses of the whole proteins and germane fragments. In each case, the molecular mass was determined by plasma desorption or laser desorption mass spectrometry and was in excellent agreement with the calculated values. Where available, the determined sequences were also in accord with the corresponding segments of the precursor molecules predicted from relevant nucleic acid sequences. Of the five, two were found to be identical (Er-2 and Er-9) and one (Er-3) was identical to a pheromone previously sequenced (Er-1), even though mating pair formation was found to take place (although to a limited extent) when cells secreting those pheromones were combined in a mixture. Comparison of the five unique sequences suggested a closer relationship between Er-1 (Er-3) and Er-10 and between Er-11 and Er-20 (44% and 56% identity, respectively) than was generally observed among the other members. This pairing was also supported by hydrophobicity analyses. Interestingly, Er-20 cannot, as a rule, induce cell union in any of the other cell types, including cells secreting Er-11, despite the fact that Er-20 and Er-11 are the most similar of the five unique sequences. Thus sequence identity and secondary structure profiles are not a good indicator of biological relatedness as manifested in heterologous receptor interaction.