A spectrophotometric method is described for the determination of microgram amounts of calcium. The procedure is extremely rapid and simple, enabling amounts of calcium in the range 2 to 3·5 µg per ml to be determined with an accuracy of about ±1 per cent. (standard deviation). The method, based on the ability of calcein (fluorescein-3,3′-bismethyliminodiacetic acid) to complex with calcium ion, measures the reduction in light absorption of a calcein solution at 506 nm on the addition of calcium.