•  The aim of the paper was to determine nitrogen compounds contributing to leaf cell osmoregulation of spinach (Spinacia oleracea) submitted to increasing salt stress. •  Sodium, free amino acids and glycine betaine contents were determined in the last fully expanded leaf of plants stressed by daily irrigation with saline water (0.17 M NaCl). •  After 20 d of treatment, when Na+ content was c. 55 umol g-1 f. wt above the control, and the reduction of stomatal conductance lowered photosynthesis to c. 60% of the control, the free amino acids of the leaves, especially glycine and serine, strongly increased. Proline and glycine betaine also increased significantly. After 27 d of treatment, when the Na+ content was c. 100 umol g-1 f. wt above the control and photosynthesis was 33% of the control, the free amino acid content, especially glycine and serine, declined. Gycine betaine, but not proline, increased further. •  Glycine betaine comprised c. 15% of the overall nitrogen osmolytes at mild salt-stress, but represented 55% of the total, when the stress became more severe. The increase of glycine betaine balanced the decline in free amino acids, mainly replacing glycine and serine (the precursors of glycine betaine) in the osmotic adjustment of the cells. Photorespiration, which increased during salt stress, was also suggested to have a role in supplying metabolites to produce compatible osmolytes.