Cytosolic free Ca 21 and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca 21 channels, as well as channel-mediated Ca 21 influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca 21 in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca 21 channel blockers La 31 or Gd 31 and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca 21 . Ca 21 fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca 21 levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca 21 increase induced by CD or CB was abolished by addition of Ca 21 channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca 21 currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca 21 -permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca 21 -permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca 21 channel activity and may consequently regulate cytoplasmic Ca 21 .The primary function of pollen and pollen tubes is to deliver sperms to egg apparatus for double fertilization that is required for sexual reproduction of flowering plants. Pollen germination and pollen tube growth are a continuous and highly polarized process characteristic of tip growth; thus pollen and pollen tubes provide an ideal model system for the study of cell polarity control and tip growth. It is well known that extracellular Ca 21 is required for pollen germination and tube growth (for review, see Steer and Steer, 1989;Taylor and Hepler, 1997;Malhó , 1998;Franklin-Tong, 1999), which indicates a possible involvement of Ca 21 influx in pollen germination and tube growth. Upon pollen hydration and germination, cytoplasmic Ca 21 concentration ([Ca 21 ] i ) at the germinal aperture where the pollen tube emerges increases to a higher level than other regions, and a tip-focused Ca 21 gradient is then established and sustained while a pollen tube grows forward (Rathore et al., 1991;Pierson et al., 1994;Feijó et al., 1995). Disruption or modification of the Ca 21 gradient inhibits pollen tube growth (Miller et al., 1992) or changes its growth direction (Malhó and Trewavas, 1996). The tip-focused Ca 21 gradient oscillates in the pollen tubes of lily (Lilium longiflorum) and other species (Holdaway-Clarke et al., 1997;Messerli and Robinson, 1997;Messerli et al., 2000). Ca 21 mobilization from intracellular Ca...