The physiochemical properties, size, shape, and surface charge, have been determined for the cytoplasmic glucocorticoid receptor complex (GR) in randomly growing HeLa S3 cells. Sucrose density gradient and gel exclusion chromatographic analysis have shown GR to undergo a marked reduction in size and shape when analyzed under increasing KCl concentration conditions. Analysis of GR prepared in a hypotonic buffer solution revealed a large, 7-8 S species with Ve/Vo ratio of 1.15, and a Stokes radius of congruent to 95 A. Increasing the KCl concentration in the analysis buffer to 0.15 resulted in reduction in GR size and shape to a 4.5 S species with the following properties: Ve/Vo = 1.38. Stokes radius = 69 A. and calculated Svedberg molecular weight = 132,000. A limit to the observed decrease in GR size and shape was obtained under hypertonic, 0.4 M KCl, conditions; a 3.75 S form was observed to elute from the gel exclusion column with a Ve/Vo = 1.40. Stokes radius = 65 A. and a calculated Svedburg molecular weight = 102,000. Exposure to higher KCl conditions, 0.6 M and 0.8 M, resulted in no further decrease in GR size or shape. Ion exchange chromatographic analysis of cytoplasmic GR revealed heterogeneous populations of GR with apparent differences in surface charge. GR binding to DEAE cellulose revealed a predominant form which eluted at 0.15 M KCl (Form I). Under both hypotonic and hypertonic conditions small populations of GR forms were observed to elute from DEAE at or approximately 0.1 M KCl (Form III), and or approximately 0.3 M KCl (Form II), respectively. Binding of GR to hydroxylapatite (HAP) confirmed the heterogenous nature of GR. Cytoplasmic GR partitioned into at least 3 forms om HAP. The predominant GR form eluted from HAP at 0.1 M K2HPO4 (Form I); secondary, and tertiary forms eluted at 0.125 M (Form II), and 0.15 M K2POH4 (Form III), respectively. Glucocorticoid receptors were observed to elute from columns of phosphocellulose, and DNA cellulose in the void volume. Two receptor forms were observed with isoelectric focusing of HeLa S3 cytoplasm in Sephadex IEF G-75 gels. The primary, and secondary species had pI values of 7.5 and 6.3, respectively. These results demonstrate that randomly growing HeLa S3 cells contain heterogeneous population of GR in the cytoplasmic compartment. Randomly growing HeLa S3 cell cytoplasm appears to contain two "unactivated" receptor species (Forms I and II) that differ in their overall surface charge properties; a third activated species (Form III) appears to arise from heat induced "activation" during cell homogenization, and is more basic in nature.