Seven of the 10 mammalian small heat shock proteins (sHSP) are expressed in muscle where they constitute 3% or more of total protein. sHSPs interact with one another, and these interactions are believed to be important for their functions. In cell types expressing multiple sHSPs, it is of interest to know which sHSPs interact with one another. We have previously shown that HSP22 interacts with itself as well as with HSP27, MKBP, and cvHSP. Using yeast two-hybrid assays and Förster resonance energy transfer microscopy, we now show that HSP22 also can interact with two additional members of the sHSP family, alphaB-crystallin and HSP20. We also show that HSP22 is found in HPLC fractions of primate cardiac muscle containing high molecular weight complexes that include alphaB-crystallin and HSP20. Our results suggest that a variety of oligomers composed of different proportions of different sHSPs may form in cell types expressing multiple sHSPs.