In teleost oocytes, yolk proteins (YP)-derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation prior to ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K⁺ ions. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. Here, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B and F (CatL, CatB and CatF), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, while CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A₁ (BA₁), a specific inhibitor of vacuolar-type H⁺-ATPase (V-ATPase), underwent maturation in vitro, but acidification of yolk globules, activation of CatB and proteolysis of YPs were prevented. In addition, MIS plus BA₁-treated oocytes accumulated less K⁺ than those stimulated with MIS alone; hence oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a V-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.