Nodulin 26 is an symbiosome membrane protein of soybean nodules that shows ion channel activity in planar lipid bilayers. Serine 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase. To study the effects of phosphorylation, nodulin 26 with Ser, Ala, or Asp at position 262 were expressed in Escherichia coli. The expressed protein possessed a histidine-rich leader sequence for purification by Ni2+ chelate fast protein liquid chromatography. Upon reconstitution into planar lipid bilayers, the recombinant proteins showed a large single channel conductance (3.1 nanosiemens (nS) in cis0.2M/trans1.0 M KCl and 1.6 nS in cis 0.2M/trans0.2 M KCl) and weak anion selectivity, similar to native soybean nodulin 26. Nodulin 26 with Ser- or Ala-262 occupied the maximal open conductance state greater than 97% of the time (3.1 nS in cis0.2M/trans1.0 M KCl) regardless of applied voltage. However, nodulin 26 with Asp-262 showed increased gating and preferential occupancy of lower subconductance states (1.8 and 0.6 nS in cis0.2M/trans1.0 M KCl) at high applied voltages (e.g. 70 mV). In situ phosphorylation of Ser-262 of nodulin 26 by calmodulin-like domain protein kinase also resulted in increased voltage-dependent gating and preferential occupancy of lower subconductance states. These results suggest that phosphorylation of serine 262 of nodulin 26 modulates channel activity by conferring voltage sensitivity.