The expression of the cystatin C gene was studied by Northern analysis of RNA isolated from the choroid plexus, other brain tissues, and liver from 11 mammalian and 4 avian species. The probe used for hybridization was cystatin C cDNA isolated previously from a rat choroid plexus cDNA library. Strong conservation of the expression of the cystatin C gene in choroid plexus was suggested by the observation of substantial levels of cystatin C mRNA in choroid plexus RNA from all mammalian and avian species studied. In contrast, levels of cystatin C mRNA in total liver RNA varied widely for mammalian as well as for avian species. It was concluded that the synthesis of cystatin C in choroid plexus has probably been conserved since the stage of the stem reptiles, the common ancestors of mammals and birds. The cystatin C gene was also found to be expressed early in ontogeny, as indicated by the observation of similar cystatin C mRNA levels in choroid plexus RNA from newly hatched and adult chickens.