Substance-dependent patients automatically and involuntarily allocate their attention to drug cues in the environment, a process referred to as attentional bias. Attentional bias is increased during periods of subjective craving and predictive of treatment outcome and relapse in substance dependence. Despite recent theoretical and clinical advances with regard to attentional bias, the underlying neurobiological mechanisms are largely unknown. The objective of the current study was to investigate the neural substrate of attentional bias and associated subjective craving in smokers. A group of smokers (n=20) and a group of age- and gender-matched nonsmoking controls (n=22) were recruited from the general population and participated in a single session of fMRI scanning while attentional processes were manipulated. Main outcome measures were blood oxygen level-dependent (BOLD) fMRI activation during an attentional bias paradigm and self reported cigarette craving. Results of the current study show that the dorsal anterior cingulate cortex, the superior parietal gyrus, and the superior temporal gyrus were more strongly activated in smokers, as compared to controls, when they had to pay attention to task-relevant information (line counting) while smoking cues were present as distracters (attentional bias). Subjective craving measures during attentional bias correlated with brain activation in the insula and putamen. To our knowledge, this is the first controlled study that shows the brain regions involved in attentional bias in smokers. The current study demonstrates that brain regions contributing to top-down attentional processing are implicated in attentional bias in smokers, suggesting that smokers have to employ more attentional resources to focus on a standard cognitive task when smoking cues are present.