Little attention has been given to the effect of interactions between different cryogenic parameters on the viability of cryopreserved algae. Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen by two-step cooling, and interactions were tested for optimizing the cooling protocol and improving freeze-tolerance. UNOVA of a general linear model suggested that interactions between both cooling rate and holding time and between holding temperature and holding time significantly affected the survival of thawed gametophytes. Based on the magnitude of effect, the importance order of factors was found to be: holding temperature, holding time, cooling rate, cooling rate x holding temperature, holding temperature x holding time. UNOVA also suggested significant main effects of pre-culture conditions and sex on survival of thawed gametophytes. Under the optimal procedure, post-thaw survivals obtained were 84 percent for male and 69 percent for female gametophytes. Male gametophytes were observed to be more tolerant to the whole procedure of cryopreservation than females. Following thawing viable gametophytes could grow asexually or give rise to morphologically normal sporophytes.