The N-terminal glycine of transducin alpha subunits is acylated by lauroyl (C12:0), myristoyl (C14:0), (cis-delta5)-tetradecaenoyl (C14:1) or (cis,cis-delta5,delta8)-tetradecadienoyl (C14:2) fatty acyl groups. We examined functional heterogeneity of transducin by sequentially eluting it from bleached outer segments using increasing concentrations of GTP then identifying the N-terminal acyl groups on the eluted alpha subunits. C14:2 acylated transducin eluted at low GTP concentrations followed by C12:0, C14:1 and C14:0 transducin at higher GTP concentrations. This suggests functional heterogeneity in the different forms of transducin alpha subunits.