Previous studies have indicated that the connective tissue cells in dermis are involved in control of interstitial fluid pressure (P_if). The aim of this study was to develop and characterize an in vitro model, representative of loose connective tissue, to study dynamic changes in fluid pressure (P_f) over a time-course of a few minutes. P_f was measured using micropipettes in cell aggregates of human dermal fibroblasts. P_f was measured in fibroblast cell aggregates which varied in size (<100 µm and >100 µm in diameter), age (day1-4) that were kept at different temperatures (approximately15, 25 and 35 °C. Pressures were measured at different depths of micropipette penetration and following treatment with Prostaglandin E1 isopropyl ester (PGE₁), Latanoprost (PGF₂) and Ouabain. P_f was positive (Pf>+2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs day1), temperature and depth of penetration of the micropipette. P_f decreased from 2.9 mmHg to 2.0 mmHg during the first 10 min after applying 10 µl of 1 mM PGE₁ (P<0.001). P_f increased from 3.0 mmHg to 4.8 mmHg (P<0.01) after administration of 10 µl of 1.4 µM Ouabain and from 3.1 mmHg to 4.4 mmHg after the addition of 5 µl of 1.42 mM PGF₂ (P> 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements having the advantage of allowing reliable and rapid screening of substances that have a potential to modify P_f as well as to study in more detail specific cell types involved in control of P_f. This study also provides evidence that fibroblasts in the connective tissue can actively modulate P_f.