Careful analysis of affinity-purified class II molecules (Ia Ag) from the murine MHC revealed the existence of a set of associated molecules that consistently co-purified with the Ia Ag. SDS-PAGE revealed that molecules of M(r) of 41 to 43 kDa and 56 to 58 kDa were associated with the affinity-purified I-A(k) Ag from the AKR B cell lymphoma AKTB-1b. Two-dimensional electrophoresis (IEF vs SDS-PAGE) allowed further characterization of four molecules in the 41- to 43-kDa range and two in the 56- to 58-kDa range. All co-purifying proteins had isoelectric points between 5.2 and 6.2. The specificity of the association of the co-purifying molecules with the I-A(k) Ag was established by using two criteria. First, with the exception of actin, proteins co-purifying with the I-A(k) molecule were not found in samples of affinity-purified class I (H-2K(k)) Ag or membrane Ig from the AKTB-1b lymphoma. Second, the use of the amino group-reactive homobifunctional cross-linker 3,3'-dithiobisproprionimidate with crude membranes from AKTB-1b increased the relative amount of materials co-purifying with I-A(k). The use of the membrane-impermeant cross-linker 3,3'-dithiobis(sulfosuccinimidyl)proprionate provided evidence that the interaction between I-A(k) and one or more of the co-purifying components occurs on the cytoplasmic face of the membrane. Two of the co-purifying molecules have been identified. The major material in the 41- to 43-kDa range was partially sequenced, leading to its identification as cytoplasmic actin. One of the components in the 56- to 58-kDa range was tentatively identified as one of the isozymes (R(II)) of the regulatory subunit of the cAMP-dependent protein kinase, based on the use of the photoaffinity label 8-azido-cAMP.