The defining feature of the life cycle in monogonont rotifers such as Brachionus plicatilis (Muller) is alternation of asexual and sexual reproduction (mixis). Why sex is maintained in such life cycles is an important unsolved evolutionary question and one especially amenable to experimental analysis. Mixis is induced by a chemical signal produced by the rotifers which accumulates to threshold levels at high population densities. The chemical features of this signal were characterized using size exclusion, enzymatic degradation, protease protection assays, selective binding to anion ion exchange and C3 reversed phase HPLC columns, and the sequence of 17 N-terminal amino acids. These studies were carried out over two years beginning in 2003 using B. plicatilis Russian strain. When rotifer-conditioned medium was treated with proteinase K, its mixis-inducing ability was reduced by 70%. Proteinase K was added to medium auto-conditioned by 1 female ml⁻¹ where typically 17% of daughters became mictic and mixis was reduced to 1%. A cocktail of protease inhibitors added to conditioned medium significantly reduced degradation of the mixis signal by natural proteases. Conditioned medium subjected to ultrafiltration retained mixis-inducing activity in the >10 kDa fraction, but the <10 kDa fraction had no significant activity. The putative mixis signal bound to an anion exchange column, eluting off at 0.72 M NaCl. These fractions were further separated on a C3 reversed phase HPLC column and mixis-inducing activity was associated with a 39 kDa protein. Seventeen amino acids from the N-terminus have strong similarity to a steroidogenesis-inducing protein isolated from human ovarian follicular fluid. The 39 kDa protein is an excellent candidate for the rotifer mixis induction signal.