Kainate-type ionotropic glutamate receptors (iGluRs) assemble primarily as heteromeric complexes at glutamatergic synapses. Despite this, little is known about their basic biophysical and structural properties. Here, we investigate the relationship between the functional behavior of recombinant heteromeric GluK2/GluK5 kainate receptors (KARs) and their subunit stoichiometry. Using outside-out patch electrophysiology, we observed that homo- and heteromeric receptors differed in their responsiveness to the agonist, AMPA, and had differing decay kinetics in response to brief applications of L-glutamate. Interestingly, the association between these two features was positively correlated with the degree of GluK2/GluK5 heteromerization. To elucidate whether this relationship is due to a fixed or variable subunit copy number, we interrogated stoichiometry directly by using single-molecule fluorescent step detection. We have made significant modifications to the original method which was based upon expressing GFP-tagged subunits in Xenopus laevis oocytes and manually counting the number of step-wise changes in fluorescence intensity due to photobleaching (Ulbrich and Isacoff, 2007). Our new approach has adapted the technique for mammalian cells and we have also eliminated user-bias by developing a fully-automated single-molecule fluorescence counting program which we have called Progressive Idealization and Filtering or PIF. Using this approach, the tetrameric and pentameric stoichiometry of homomeric GluK2 (n=1312 spots) and α1 glycine receptors (n=1186 spots) respectively were correctly identified. In both cases, the GFP maturation rate (pm) was determined to be 53%, indicating that maturation is fluorophore-dependent. Interestingly, best fits of the data were obtained by assuming a single photobleaching spot could contain two protein complexes. Applying this same method to heteromeric GluK2/GluK5 KARs revealed that these subunits assemble with a fixed stoichiometry of 2:2 (n=1659 spots). Similar to previous results, pm was 53% and best fits of the data were obtained by assuming multiple receptor complexes could occupy a single resolvable spot. Since prior work on NMDA-type iGluRs has shown that they are also obligate heteromers, our work suggests that this may be a common feature of all heteromeric iGluRs.