We have measured the distribution of cytoplasmic calcium in lily pollen tubes by microinjecting them with indo-1 and performing fluorescence ratio image analysis on them. All of the 16 tubes that were growing at the time of the calcium measurements showed a gradient of [Ca2+]i in the tip region, with Ca2+ being 1.25 to 3.32 times higher at the distal end in 15 cases and more than 5 times higher in one case. The extent of the gradient ranged from 22 to 65 microns. Most of the 15 nongrowing tubes either had no gradient or had lower Ca2+ in the tip region. While we have confirmed a previous report that lily pollen tubes can be loaded with the membrane-permeable acetoxymethyl ester forms of calcium indicators, the dyes loaded in this way are visibly partitioned into organelles and this method of loading is, therefore, not useful for the measurement of [Ca2+]i. Iontophoresis of the dye free acids into tubes produces a more uniform and diffuse fluorescence which does not appear to partition into organelles. Indo-1 remains in the pollen tubes longer than fura-2. The correlation between growth and the [Ca2+]i gradient in the apical portion of the pollen tube is discussed in relation to previous reports that have suggested that such a gradient should exist during polarized growth.