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Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, α (p92) and β (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3α is deleted in Stat3β and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3α and Stat3β into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3α or GFP-Stat3β revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3α or GFP-Stat3β. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3α, it was >180 mm for GFP-Stat3β. Furthermore, although the intranuclear mobility of GFP-Stat3α was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3β in unstimulated cells was slower than that of GFP-Stat3α in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3α and Stat3β have distinct intracellular dynamics, with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3β. [ABSTRACT FROM AUTHOR]