The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4). This is a conditional mutant, resembling the wild type at the permissive temperature (?20°C) and expressing mutant phenotypes at the restrictive temperature (?30°C). Root cells in rsw4 at the restrictive temperature undergo non-disjunction and other indications of the loss of separase function. To determine to what extent separase activity remains at 30°C, we examined the effect of the mutation on meiosis, where the effects of loss of separase activity through RNA interference are known; and in addition, we examined female gametophyte development. Here, we report that, at the restrictive temperature, replicated chromosomes in rsw4 meiocytes typically fail to disjoin and the cohesin complex remains at centromeres after metaphase. Meiotic spindles appear normal in rsw4 male meiocytes; however the mutation disrupts the radial microtubule system, which is replaced by asymmetric arrays. Surprisingly, female gametophyte development was relatively insensitive to loss of separase activity, through either rsw4 or RNAi. These effects confirm that phenotypes in rsw4 result from loss of separase activity and establish a role for separase in regulating cell polarization following male meiosis.