AbstractHigh mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG‐14 and ‐17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full‐length masses demonstrated mono‐, di‐, and triphosphorylation of HMG‐14 and mono‐ and diphosphorylation of HMG‐17 from OA treated cells, whereas HMG‐14 and ‐17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG‐17, and Ser20 and Ser24 in HMG‐14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG‐14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG‐14 and ‐17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG‐14 and ‐7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei.