Whole-body and 20 individual-tissue (51)Cr-RBC (red cell space; RCS) and (99)Tc-diethylenetriaminepentaacetic acid (extracellular space; ECS) spaces were measured in seven unanesthetized Pacific hagfish (Eptatretus stouti). Volume indicators were administered via a dorsal aortic cannula implanted the previous day. Blood samples were collected at 6, 12, 18, and 24 h after injection. Tissues were removed at 24 h and radioactivity was measured; tissue water content (percent of wet weight) was determined by desiccation at 95 degrees C for 48 h. Mixing rates of both indicators were identical and were essentially complete by 12 h, indicating that blood convection is the rate-limiting process. At 24 h, the whole-body RCS was 19.3+/-2.1 mL kg(-1) body weight, and the ECS was 338.5+/-15.2 mL kg(-1) body weight. Blood volume estimated from the 24-h RCS and the mean central hematocrit (14%) was 137.9 mL kg(-1) body weight. Liver RCS (118.6+/-30.5 microL g(-1) tissue weight) was twice that of any other tissue and was also the most variable, ranging from 59 to 263 microL g(-1), whereas liver ECS (406.0+/-34.3 microL g(-1)) was in the range of other tissues, and water content (66.9%+/-3.5%) was low. Gill RCS (55.9+/-5.7 microL g(-1)), ECS (415.3+/-37.7 microL g(-1)), and percent water (83.1%+/-0.8%) were higher than most other tissues. RCS, ECS, and percent water were consistently lowest in ovum (1.1+/-0.02 microL g(-1), 111.1+/-4.3 microL g(-1), 51.3%+/-3.5%, respectively). Tongue, notocord, and myotome had generally lower RCS (2.1+/-0.4, 2.2+/-0.5, 7.1+/-0.1 microL g(-1), respectively) and ECS (121.2+/-7.0, 246.3+/-17.4, 185.3+/-16.7 microL g(-1), respectively), although their water content was in the midrange (74.7+/-0.5, 81.2+/-1.6, 74.4%+/-0.6%, respectively). Skin had a low RCS (6.8+/-1.1) and midrange ECS (387.5+/-28.0) but very low water content (61.2%+/-2.1%). These findings confirm that hagfish blood volume is at least twice as large as other fish, whereas our estimate of extracellular fluid volume is larger than previously reported and more in line with the predicted interstitial volume. RCS, ECS, and water content vary, often independently, between tissues, which may perhaps be indicative of specific tissue needs or functions. A distinct spleen is lacking in hagfish, and the liver appears to serve this function by sequestering red cells. To our knowledge, this is the first report of tissue ECS in Myxiniformes.