GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Galpha(i/o)-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Galpha(o)- and Galpha(i)-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Galpha(i1), Galpha(i2), and Galpha(i3) and Galpha(o). Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to Galpha(i1)-, Galpha(o)-, and Galpha(s)-subunits was assessed by surface plasmon resonance; all of the motifs bound Galpha(i1), but exhibited low affinity towards Galpha(o). GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Galpha(i1), Galpha(i2), and Galpha(i3). Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Galpha(o) was significantly lower than that toward Galpha(i1), suggesting that the in vivo targets of GPSM2 are most likely to be Galpha(i)-subunits.