In functional brain imaging there is controversy over which hemodynamic signal best represents neural activity. Intrinsic signal optical imaging (ISOI) suggests that the best signal is the early darkening observed at wavelengths absorbed preferentially by deoxyhemoglobin (HbR). It is assumed that this darkening or "initial dip" reports local conversion of oxyhemoglobin (HbO) to HbR, i.e., oxygen consumption caused by local neural activity, thus giving the most specific measure of such activity. The blood volume signal, by contrast, is believed to be more delayed and less specific. Here, we used multiwavelength ISOI to simultaneously map oxygenation and blood volume [i.e., total hemoglobin (HbT)] in primary visual cortex (V1) of the alert macaque. We found that the hemodynamic ''point spread,'' i.e., impulse response to a minimal visual stimulus, was as rapid and retinotopically specific when imaged by using blood volume as when using the initial dip. Quantitative separation of the imaged signal into HbR, HbO, and HbT showed, moreover, that the initial dip was dominated by a fast local increase in HbT, with no increase in HbR. We found only a delayed HbR decrease that was broader in retinotopic spread than HbO or HbT. Further, we show that the multiphasic time course of typical ISOI signals and the strength of the initial dip may reflect the temporal interplay of monophasic HbO, HbR, and HbT signals. Characterizing the hemodynamic response is important for understanding neurovascular coupling and elucidating the physiological basis of imaging techniques such as fMRI.fMRI ͉ imaging ͉ macaque ͉ visual ͉ neurovascular coupling C erebral hemodynamics respond quickly and specifically to local neural activity (1, 2). Hemodynamic signals are thus used extensively as proxies for such activity in functional neuroimaging techniques like fMRI and intrinsic signal optical imaging (ISOI). There has been considerable debate, however, as to which of the possible hemodynamic signals, e.g., changes in local blood oxygenation, volume, or flow, with their distinct response properties, constitutes the ''best'' signal for inferring neural activity (1,(3)(4)(5).The debate regarding the best signal sharpened with reports of initial dips in both ISOI and fMRI signals. ISOI studies consistently found a brief stimulus-evoked darkening followed by a strong brightening at imaging wavelengths preferentially absorbed in deoxyhemoglobin (HbR) [e.g., at 605 nm (6)]. The darkening, later termed the initial dip*, was interpreted as a local conversion of oxyhemoglobin (HbO) to HbR caused by increased oxygen consumption by local neurons before any active vascular response (1, 7) The subsequent brightening was taken to measure the ''rebound'' in [HbO] † caused by a delayed, stimulus-triggered increase in cerebral blood flow (7).In parallel, some fMRI studies reported finding an initial dip before the rise in the blood oxygen level-dependent (BOLD) signal (8). Because the BOLD signal measures changes in [HbR] alone ‡ , the fMRI initial dip was seen a...