Although intratympanic (IT) administration of drugs has gained wide clinical acceptance, the distribution of drugs in the inner ear following IT administration is not well established. Gadolinium (Gd) has been previously used as a marker in conjunction with magnetic resonance imaging (MRI) to visualize distribution in inner ear fluids in a qualitative manner. In the present study, we applied gadolinium chelated with diethylenetriamine penta-acetic acid (Gd-DTPA) to the round window niche of 12 guinea pigs using Seprapack(TM) (carboxlmethylcellulose-hyaluronic acid) pledgets which stabilized the fluid volume in the round window niche. Gd-DTPA distribution was monitored sequentially with time following application. Distribution in normal, unperforated ears was compared with ears that had undergone a cochleostomy in the basal turn of scala tympani and implantation with a silastic electrode. Results were quantified using image analysis software. In all animals, Gd-DTPA was seen in the lower basal scala tympani (ST), scala vestibuli (SV), and throughout the vestibule and semi-circular canals by 1 h after application. Although Gd-DTPA levels in ST were higher than those in the vestibule in a few ears, the majority showed higher Gd-DTPA levels in the vestibule than ST at both early and later time points. Quantitative computer simulations of the experiment, taking into account the larger volume of the vestibule compared to scala tympani, suggest most Gd-DTPA (up to 90%) entered the vestibule directly in the vicinity of the stapes rather than indirectly through the round window membrane and ST. Gd-DTPA levels were minimally affected by the implantation procedure after 1 h. Gd-DTPA levels in the basal turn of scala tympani were lower in implanted animals, but the difference compared to non-implanted ears did not reach statistical significance.