A tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungusNeurospora crassa. The Ca2+gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+gradients, but this can be explained by Ca2+diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+channels generate and maintain the tip-high calcium gradient.