Acyl carrier protein plays an essential role in bacterial fatty acid synthesis and has been recognized as an attractive new antibiotic target. In a previous study using TAP tagged ACP, we identified two ACP binding partners not directly involved in lipid metabolism, AidB and SpoT, that showed increased binding to ACP in stationary phase. In the current project, we found that the increase in binding of SpoT, a stringent response protein, to ACP appears to be due to increased affinity. In contrast, the increase in binding of AidB, an adaptive response protein, appears to be due to increased expression of AidB. Transformation of SpoT-TAP strains with pGEX-ACP vectors followed by purification revealed that SpoT interacts with wild type ACP, but not with ACP mutants that neutralized helix II or cannot fold properly. We used a far western approach to confirm the direct interaction between ACP and AidB in vitro. Overall, these results support the hypothesis that interaction of ACP with SpoT is highly specific and may reflect a regulatory role for ACP, while that with AidB might reflect non-specific interactions.