Heat shock proteins of the 70 kDa family (HSP70) are intracellular chaperones necessary forthe cell to maintain protein homeostasis. In the cytosol, under normal conditions, these proteins promotethe correct folding of proteins, preventing their aggregation, and are involved in protein transport and cellsurvival. Among the HSP70, there is a pool of stress-inducible proteins Hsp70, which significantly increases inresponse to a number of stress factors and facilitates cell recovery after stress. Tumor cells, unlike normal, arecharacterized by the ability to present Hsp70 on the surface of the cell membrane. Membrane-bound Hsp70can be considered as a danger signal and enhance or inhibit immune responses. A three-dimensional modelof cells in the spheroids in varying degrees simulates the structural organization of solid tumors. In cultures ofmulticellular spheroids (3D), hypoxia and nutrient gradients are formed within the spheroids, which can affectthe translocation of Hsp70 to the cell membrane. The purpose of this work was a comparative analysis of Hsp70expression on tumor cells of various origins when cultivated in a monolayer state (2D) and 3D cultures. Analysiswas carried out on breast and pancreatic tumor cell lines, colon and prostate carcinomas, and lymphomas usingflow cytometry and confocal microscopy methods. Cultivation in 3D cultures was performed using the anti-adhesive PolyHEMA substrate. The results showed that not all carcinomas from our panel express Hsp70 inboth 2D and 3D cultures. Some tumor lines have membrane Hsp70 only in 3D cultures. Hsp70 expression wasdetected on: BT20 breast cancer cells; colon carcinoma SW837; pancreas PANC1; and prostate PC-3. Analysisof Hsp70-positive carcinomas of various localizations in 2D and 3D models may be useful for the applicationof antibodies against Hsp70 as a vector for the delivery of anticancer drugs