The calpain-like protease from Allomyces arbuscula was phosphorylated in vitro by a serine/threonine protein kinase from Neurospora crassa typical of casein kinase II. The protease contained covalently bound phosphate when the culture was labelled in vivo with P-32i. The phosphoryl linkage was sensitive to alkaline phosphatase. Phosphoserine was the major phosphohydroxylamino acid detected after acid hydrolysis of the in vitro and in vivo phosphorylated enzyme. Phosphopeptide maps of the protease phosphorylated either in vitro or in vivo were similar. These data suggest that in vivo the protease may be regulated by phosphorylation-dephosphorylation mechanisms.