Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have shown that it may be involved in the formation of coated vesicles. In this report, three different alternatively spliced dynamin-like protein variants (DLP1-WT, -11, and -37) from rat brain were identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel rat alternatively spliced variant (DLP1-37), not described previously, was identified. We examined the interaction of these three rat brain dynamin-like protein variants with glycogen synthase kinase 3 beta (Gsk-3 beta) using the yeast two-hybrid screening, in vitro binding assay, and immunoprecipitation analysis. It was found that all three examined rat brain dynamin-like protein variants can bind to Gsk-3 beta. Moreover, in vitro kinase (phosphorylation) assay showed that mammalian dynamin-like protein acts as a substrate for glycogen synthase kinase 3 beta. These data suggest that Gsk-3 beta may participate in a functional role in dynamin-like proteins in vesicle trafficking.