We here describe the generation of novel, green fluorescent protein-based Ca(2+) indicators targeted to the peroxisome lumen. We show that (i) the Ca(2+) concentration of peroxisomes in living cells at rest is similar to that of the cytosol; (ii) increases in cytosolic Ca(2+) concentration (elicited by either Ca(2+) mobilization from stores or Ca(2+) influx through plasma membrane Ca(2+) channels) are followed by a slow rise in intraperoxisomal [Ca(2+)]; (iii) Ca(2+) influx into peroxisomes is driven neither by an ATP-dependent pump nor by membrane potential nor by a H(+)(Na(+)) gradient. The peroxisomal membrane appears to play a low pass filter role, preventing the organelle from taking up shortlasting cytosolic Ca(2+) transients but allowing equilibration of the peroxisomal luminal [Ca(2+)] with that of the cytosol during prolonged Ca(2+) increases. Thus, peroxisomes appear to be an additional cytosolic Ca(2+) buffer, but their influx and efflux mechanisms are unlike those of any other cellular organelle.