Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.