The visualization of inducible nitric oxide synthase (iNOS) in vivo with specific radioactive probes could provide a valuable insight into the diseases associated with upregulation of this enzyme. Aiming at that goal, we have synthesized a novel family of conjugates bearing a pyrazolyl-diamine chelating unit for stabilization of the fac-[M(CO)3]+ core (M = 99mTc, Re) and pendant guanidino (L1 = guanidine, L 2 = N-hydroxyguanidine, L3 = N-methylguanidine, L 4 = N-nitroguanidine) or S-methylisothiourea (L5) moieties for iNOS recognition. L1-L5 reacted with fac-[M(CO) 3(H2O)]+, yielding complexes of the type fac-[M(CO)3(k3-L)]+ (M = Re/99mTc; 1/1a, L = L1; 2/2a, L = L2; 3/3a, L = L3; 4/4a, L = L4; 5/5a, L = L5), which were fully characterized by the usual analytical methods in chemistry and radiochemistry, including X-ray diffraction analysis in the case of 1. The rhenium complexes 1-5 were prepared as "cold" surrogates of the 99mTc(I) complexes. Enzymatic assays with murine purified iNOS demonstrated that L1, L2, 1 and 2 are poor NO-producing substrates. These assays have also shown that metallation of L4 and L5 (Ki > 1000 μM) gave complexes with increased inhibitory potency (4, Ki = 257 μM; 5, Ki = 183 μM). The organometallic rhenium complexes permeate through LPS-treated RAW 264.7 macrophage cell membranes, interacting specifically with the target enzyme, as confirmed by the partial suppression of NO biosynthesis (ca. 20% in the case of 4 and 5) in this cell model. The analog 99mTc(I)-complexes 1a-5a are stable in vitro, being also able to cross cell membranes, as demonstrated by internalization studies in the same cell model with compound 4a (4h, 37 °C; 33.8% internalization). Despite not being as effective as the α-amino-acid-containing metal-complexes previously described by our group, the results reported herein have shown that similar 99mTc(I)/Re(I) organometallic complexes with pendant amidinic moieties may hold potential for targeting iNOS expression in vivo.