This study tested the hypothesis that changes in the blood concentration, and possibly in the perfusion, of different areas in the brain can be assessed by the use of ultrasound contrast agent (CA) and (linear) echo densitometry. The experiments were performed with piglets (n=3) under general anesthesia and artificial ventilation. Ultrasound CA was administered through a femoral vein as a short bolus. First passage wash-in curve was measured from image gray level during continuous low level (mechanical index<0.2) ultrasound imaging. This curve was obtained from 1-cm2 areas of the cortex (surface), the brain stem (inner) and the left carotid artery (vessel). Cerebral hemoglobin concentration changes were measured with near-infrared spectroscopy (NIRS). This approach enabled a cross-validation of these techniques. The measurements were repeated under conditions of normocapnia, mild hypercapnia and deep hypercapnia. Several physiologic signals, as well as the carotid blood flow, were measured simultaneously and related to gray level by linear regression analysis. The most significant results found were a high R2-statistic of the regression of the percentage change of the peak of the surface and inner wash-in curves with the arterial carbon dioxide pressure (R2=0.63 and R2=0.70, respectively), the blood pH (R2=0.79 and R2=0.81), the carotid flow (R2=0.75 and R2=0.72) and the partial arterial oxygen pressure (R2=0.47 and R2=0.55). Finally, a high correlation of peak gray level with total hemoglobin concentration change, independently measured by NIRS, was found (R2=0.69). In conclusion, these experiments show a reasonable intersubject variability of various relative measures derived from gray level ultrasound wash-in curves. High sensitivity to physiologic changes related to hypercapnia was observed for the peak contrast of wash-in curves. For up-slope and area-under-the-curve (first passage) this was lower but still highly significant. The gray-level ultrasound measures are highly correlated to changes in regional hemoglobin concentration in brain tissue assessed by NIRS.