The aim of our study was to find out the features of the nitric oxide synthase (NOS) isoforms expression in longitudinal and transversal layers of the myocardium of the left ventricle in male rats of 3 months, which are descendants of female rats with experimental gestational diabetes (EGD). Materials and methods. Study was carried out on 10 male rats descendants of female rats with normal course of pregnancy and 10 descendants of female rats with EGD. We evaluated the expression of neuronal, endothelial and inducible isoforms of NOS in histological sections both of transversal and longitudinal layers of the myocardium of left ventricle. With the aim to analyze the pattern of the NOS isoforms expression in 5 um histological slices of left ventricle myocardium we have carried out a complex of histochemical assays. Slices were allocated to 3 groups: 1 st one was incubated with rabbit IgG to neuronal NOS (Santa Cruz Biotechnology, USA) in dilution of 1:200; 2 nd group underwent the incubation with rabbit IgG to endothelial NOS (eNos) (Santa Cruz Biotechnology, USA) in dilution 1:200; 3 rd group was incubated with mice IgG to inducible NOS (iNos) conjugated with FITC (Santa Cruz Biotechnology, USA) in dilution of 1:200. The analysis of images was carried out with VIDAS-2.5 application package (Kontron Elektronik, Germany). Microimages of the left ventricle myocardium obtained with AxioScope (Carl Zeiss, Germany) and COHU 4922 (COHU Inc., USA) camera were processed in the system of digital image analysis VIDAS-386 (Kontron Elektronik, Germany). Results. In the study we have found that prenatal hyperglycaemia leads to the significant changes of the expression of NOS isoforms in the myocardium of left ventricle in male rats descendants of females with EGD, and the contain and the allocation of these enzymes are dependent both on type of the enzyme and its location in muscular layers. For eNOS the increase of the expression and the allocation in both transversal and longitudinal layers was typical, and for nNOS and iNOS we have found the significant decrease of the expression in transversal layers and the significant increase in longitudinal layers. Conclusion. We believe, the glucose homeostasis violation in foetus during the last trimester of the pregnancy changes the ratio of the NOS isoforms expression in the left ventricle myocardium and affects the balance of them in different layers, which could be a reason of the cardiovascular pathology development in adulthood.