The protein NaGSL1 (Nicotiana alata glucan synthase-like 1) is implicated in the synthesis of callose, the 1,3-β-glucan that is the major polysaccharide in the walls of N. alata (flowering tobacco) pollen tubes. Here we examine the production, intracellular location and post-translational processing of NaGSL1, and relate each of these to the control of pollen-tube callose synthase (CalS). The 220 kDa NaGSL1 polypeptide is produced after pollen-tube germination and accumulates during pollen-tube growth, as does CalS. A combination of membrane fractionation and immunoelectron microscopy revealed that NaGSL1 was present predominantly in the endoplasmic reticulum and Golgi membranes in younger pollen tubes when CalS was mostly in an inactive (latent) form. In later stages of pollen-tube growth, when CalS was present in both latent and active forms, a greater proportion of NaGSL1 was in intracellular vesicles and the plasma membrane, the latter location being consistent with direct deposition of callose into the wall. N. alata CalS is activated in vitro by the proteolytic enzyme trypsin and the detergent CHAPS, but in neither case was activation associated with a detectable change in the molecular mass of the NaGSL1 polypeptide. NaGSL1 may thus either be activated by the removal of a few amino acids or by the removal of another protein that inhibits NaGSL1. These findings are discussed in relation to the control of callose biosynthesis during pollen germination and pollen-tube growth.