The aim of the present study was to quantify osmotic pressures directly in the translocation pathway, from leaf to growing root tip, in order to understand the forces driving solutes from a source to a sink. Solutes move through the translocation pathway down an osmotically derived turgor gradient. Accordingly aphid stylectomy and single cell sampling techniques have been combined to examine the osmotic pressure of root phloem and growing root cells. Sieve tube sap was obtained from shoots and, for the first time, roots of barley seedlings using aphid stylectomy. Vacuolar sap was also obtained from a variety of cells in leaf and root tissues using single cell sampling methods. Osmotic pressure of sieve tube sap from roots and shoots was measured at high temporal resolution (within min) and over long periods of time (up to 24 h). Osmotic pressure did not change significantly in the minutes immediately following excision, suggesting that confidence can be placed in the assumption that stylet exudate is representative of sieve tube sap in vivo. There were no differences in the osmotic pressure of sieve tube sap from shoots (1.24±0.26 MPa, n = 10) or roots (1.42±0.15 MPa, n = 13). However, osmotic pressure of sap from root cortical cells (0.71±0.09, n = 12) was about 0.7 MPa lower than that of the sieve elements from roots, this difference may be maintained by consumption of incoming solutes at the root tip. Results are discussed in the context of pressure driven flow in the phloem and symplastic contact between root tip cells and sieve tube. It is hoped that the approach described here will provide important insights into the nature of the relationship between root cell extension and assimilate supply through the phloem.