Cyclin-dependent kinases regulatory subunit 2 (CKS2) is overexpressed and associated with aggressiveness of several cancers. The reason for this is not clarified. CKS2 is known to bind the cell cycle regulatory proteins cyclin-dependent kinase 1 and 2 (CDK2 and CDK2) and mitochondrial single-stranded DNA-binding protein (SSBP1). SSBP1 participates in the biogenesis of mitochondria. These interactions have not previously been explored by in situ proximity ligation assay (PLA). The purpose of this study was to investigate cellular localization of CKS2 in HeLa cells. Further, co-localization and interactions of CKS2 with CDK1, CDK2 and SSBP1 were explored by immunofluorescence cytochemistry and PLA. Several fixation methods were tested for optimization of the PLA. Immunofluorescence cytochemistry showed that CKS2 was distributed in large foci in the nucleus and in small foci in the cytoplasm. In addition, CKS2 and CDK1 were localized at the centrosomes both in interphase and mitosis. The nuclear CKS2 foci were mainly restricted to weakly Hoechst stained areas, and they were co-localized with CDK1 and CDK2. Co-localization between CKS2, CDK1 and CDK2 was also apparent in the cytoplasm in interphase and mitosis. A small part of the cytoplasmic CKS2 and CDK1 foci co-localized with a SSBP1. PLA functioned well for both formalin and methanol fixated cells. However, formalin fixated cells required treatment with an extra reagent, e.g. a detergent buffer or methanol for production of PLA signals in the nucleus. Methanol fixation alone produced reproducible nuclear signals and was used for the examination of CKS2-CDK interactions, whereas combined formalin and methanol fixation were used for studying CKS2-SSBP1 and CDK1-SSBP1 interactions. PLA confirmed that CKS2 interacted with CDK1 and CDK2 in the nucleus and with CDK1, CDK2 and SSBP1 in the cytoplasm. The distribution of the CDK1-SSBP1 interactions in the cytoplasm resembled that of the CKS2-SSBP1 interactions. The results indicated that the large CKS2 foci in the nucleus were localized in euchromatin, which is transcriptionally competent DNA, and thus support the hypothesis that CKS2 has a role in the transcription of DNA. CKS2 probably interacted with CDK1 and CDK2 in these areas. Some of the cytoplasmic CKS2 and CDK1 foci interacted with SSBP1, probably within the mitochondria. In addition, the co-localization of CKS2 and CDK1 at the centrosomes lends support to the hypothesis of CKS-dependent activation and inactivation of the CDK1-CCNB complex prior to and in the mitosis.