The Tat (twin-arginine translocation) pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the mycelial soil-dwelling bacterium Streptomyces. We recently examined the localization of Tat components (TatABC) in time-lapse imaging and demonstrated that all three components colocalize dynamically with a preference for apical sites. Here we apply an in-house single particle tracking package to quantitatively analyze the movement of the TatA subunit, the most abundant of the Tat components. Segmentation and analysis of trajectories revealed that TatA transitions from free to confined movement and then to fixed localization. The sequence starts with a mixed punctate and dispersed localization of TatA oligomers, which then develop into a few larger still foci, and finally colocalize with TatBC to form a functional translocation system. It takes 15-30 min for the Tat export complex to assemble and most likely become active. With this study we provide the first example of quantitative analysis of dynamic protein localization in Streptomyces, which is applicable to the study of many other dynamically localizing proteins identified in these complex bacteria. (C) 2013 Elsevier Inc. All rights reserved.