AbstractEmploying a newly developed computer‐assisted system for visualizing and quantitating cell motility in three dimensions, we have examined the 3‐dimensional changes in cell shape and the dynamics of pseuodopod extension during translocation ofDictyosteliumamoebae. Amoebae exhibit a 3‐dimensional behavior cycle with an average period of 1.5 min. The cycle includes a transient pseudopod extension phase in the x, y axis followed by a z‐axis expansion phase. Anterior pseudopod extension in the x, y axis is accompanied by a decrease in height, not by uropod retraction. The increase in height is accompanied by uropod retraction. In the pseudopod extension phase in the x, y axes, pseudopods form either anteriorly or laterally, and either on or above the substratum. Pseudopods which initially form on the substratum in almost all cases continue to expand as the anterior end of the cell. In the case of lateral pseuodopods, anteriorization leads to a turn. Approximately half of anterior pseudopod and two‐thirds of lateral pseudopods which initially form above the substratum are retracted. These results suggest that pseudopod‐substratum interaction plays a fundamental role in the regulation of directionality and turning in the translocation phase of the 3‐dimensional behavior cycle. © 1994 W