To gain insight into the characteristics of organelle movement and the underlying actomyosin motility system in tobacco pollen tubes, we collected data points representing sequential organelle positions in control and cytochalasin-treated cells, and in a sample of extruded cytoplasm. These data were utilized to reconstruct approximately 900 tracks, representing individual organelle movements, and to produce a quantitative analysis of the movement properties, supported by statistical tests. Each reconstructed track appeared to be unique and to show irregularities in velocity and direction of movement. The regularity quotient was near 2 at the tip and above 3 elsewhere in the cell, indicating that movement is more vectorial in the tube area. Similarly, the progressiveness ratio showed that there were relatively more straight trajectories in the tube region than at the tip. Consistent with these data, arithmetical dissection revealed a high degree of randomlike movement in the apex, lanes with tip-directed movement along the flanks, and grain-directed movement in the center of the tube. Intercalated lanes with bidirectional movement had lower organelle velocity, suggesting that steric hindrance plays a role. The results from the movement analysis indicate that the axial arrangement of the actin filaments and performance of the actomyosin system increases from tip to base, and that the opposite polarity of the actin filaments in the peripheral (+-ends of acting filaments toward the tip) versus the central cytoplasm (+-ends of actin filaments toward to the grain) is installed within a few minutes in these tip-growing cells.